Method for treatment of blood tumor using anti-TIM-3 antibody

ABSTRACT

Disclosed is a therapeutic method including administering a TIM-3 antibody or its TIM-3 binding fragment to a subject who is suspected to be suffering from blood tumor and in whom TIM-3 has been expressed in a Lin(−)CD34(+)CD38(−) cell fraction of bone marrow or peripheral blood or a subject who has been received any treatment for blood tumor. Conceived diseases include those diseases which can be treated through the binding or targeting of the TIM-3 antibody or its TIM-3 binding fragment to blood tumor cells (AML cells, CML cells, MDS cells, ALL cells, CLL cells, multiple myeloma cells, etc.), helper T cell (e.g., Th1 cells, Th17 cells), and antigen-presenting cells (e.g., dendritic cells, monocytes, macrophages, and cells resembling to the aforementioned cells (hepatic stellate cells, osteoclasts, microglial cells, intraepidermal macrophages, dust cells (alveolar macrophages), etc)).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/136,394 filed Dec. 20, 2013 (U.S. Pat. No. 9,103,832), which is adivisional of U.S. application Ser. No. 13/263,434 filed Oct. 7, 2011(U.S. Pat. No. 8,647,623), which is a National Stage Entry ofInternational Application No. PCT/JP2010/056445 filed Apr. 9, 2010,which claims benefit of U.S. Provisional Application No. 61/168,428filed Apr. 10, 2009, the contents of all of which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an agent for treating or diagnosingmyeloid malignancy, especially acute myelogenous leukemia (AML),comprising an antibody to human TIM-3 protein (another name: humanHAVCR2) as an active ingredient. In addition, the present inventionrelates to a method for isolating leukemic stem cells.

BACKGROUND ART

Regarding Malignant Tumor:

A malignant tumor (cancer) is the first leading cause of death in Japanand the number of patients is increasing every year, and the developmentof a drug and a therapeutic method having high efficacy and safety isstrongly desired. Examples of the cause of forming a malignant tumorinclude a mutation of DNA caused by radiation, ultraviolet rays andvarious carcinogenic substances. Studies on malignant tumors have beenfocused on identification of these genetic changes molecularbiologically. As a result, it is considered that tumorigenictransformation is induced by accumulation of a large number of mutationsand the like. It has been shown by a cell line model and the like thatsome decisive mutations directly connected with the tumorigenictransformation. Regarding leukemia as one of the objective diseases ofthe invention, many chromosomal abnormalities have been identified andclassified. In many of the case, translocation of chromosome is foundand the translocation associated genes have already been identified inthe main translocation of chromosome. By functional analyses of thetranslocation related genes, a case has been found that these genesrelates to the onset of leukemia.

Regarding Cancer Stem Cell:

On the other hand, a so-called cancer stem cell hypothesis has beenproposed for a long time from the viewpoint of cell biology, statingthat stem cell is the origin of a malignant tumor similar to the normaltissue. The stem cell is defined as a cell having self-renewal capacityand pluripotency and generally divided roughly into totipotency stemcell and tissue stem cell. The tissue stem cell is origin of specifictissues and organs such as of blood system, liver, nerve system and thelike and present at an extremely low frequency. Among them, the study ofhematopoietic stem cell is at the most advanced stage. It has beenreported that a hematopoietic system can be reconstituted over a longperiod of time by transplanting one hematopoietic stem cell into a mousein which the hematopoietic system was destructed by a lethal dose ofirradiation (Non-patent Reference 1). Different from the normal stemcell, studies on cancer stem cells have been delayed for a long timesince their true nature could not been found. However, a cancer stemcell has been identified for the first time in acute myelocyticleukemia, in 1997 by Dick et al (Non-patent Reference 2). Thereafter,the presence of cancer stem cells was reported in various malignanttumors. In summing up, cancer stem cells are present at a frequency ofseveral % or less of the whole tumor and the presence of them are rareas well as normal stem cells. It is considered that the remaining cellswhich form the tumor are tumor precursor cells in which proliferationability is limited or tumor cells.

From these reports, it was shown that hierarchy is present even in tumoras well as the normal tissue, and the cancer stem cell residing at thistop (origin) has strong tumor forming ability. Based on the above, it isconsidered that the beginning of the onset of malignant tumors is achange from a normal stem cell to a so-called cancer stem cell byaddition of several mutations.

Characteristics and Therapeutic Problems of Cancer Stem Cells:

In summing up many reports, it is considered that cancer stem cells aremaintaining various characteristics possessed by the normal stem cells.Examples of similarities include rarity of the cell, a microenvironment(niche) in which the stem cells exist, expression of a multiple drugresistance gene, cell cycle, and the like.

Particularly, the characteristics that cancer stem cells express a groupof multiple drug resistance genes and are at the stationary phase ofcell cycle similar to the normal stem cells could become atherapeutically great problem. A multiple drug resistance gene BCRP is apump which impairs the drug efficacy by discharging various antitumoragents into outside of cells, and a method for collecting stem cellsmaking use of the activity has been reported (Non-patent Reference 3).In addition, the stem cell is under a state of “hibernation” in order tokeep providing cells for its whole life (Non-patent Reference 4) and itreduces in sensitivity for many antitumor agents and radiation(Non-patent References 5 and 6). Based on the above characteristics, itis considered that the rare cancer stem cell which shows resistance tothe therapy is a cause of tumor recurrence.

Regarding Molecular Target Drug:

Three main courses of the treatment of a malignant tumor includeantitumor agent therapy, radiation therapy and excision. Treatment forthe blood tumor is limited to the antitumor agent therapy and radiationtherapy, and as described in the above, the cancer stem cell can have aresistance to these treatments. Another problem is that side effects arelarge since these two treatments affect the entire body. It is amolecular target drug that is expected as a resolving means for thisproblem. It has a possibility to reduce side effects by exhibiting itsdrug efficacy only in the cell expressing the target molecule.

Examples of typical drugs of the molecular target drug in the field ofblood diseases include imatinib and rituximab. Imatinib targets at aleukemia-causing factor called Bcr-Abl produced by a chromosomalabnormality (Philadelphia chromosome) which is observed in 95% ofchronic myeloid leukemia (CML) patients. Imatinib is a low molecularweight drug which induces suicide of leukemia cell by inhibitingfunction of Bcr-Abl. Rituximab is a therapeutic antibody whichrecognizes CD20 as a surface molecule on a B cell and has an antitumoreffect on non-Hodgkin lymphoma, a malignant tumor of B cell. On theother hand, molecular target drugs for AML are few, and there is only anagent gemtuzumab ozogamicin (Mylotarg) in which an antibioticcalicheamicin is bound to a monoclonal antibody to CD33 known as an AMLcell surface antigen (Non-patent Reference 7). However, it can be saidthat that the use of Mylotarg is limited since Mylotarg can be appliedonly when a patient meets the following four limitations such as theexpression rate of CD33 of 80% or more, a case of recurrency, age of 60or more, and resistance to other chemical therapy. Based on the above,it can be said that discovery of a new target gene and development of atherapeutic agent for this are important inventions which directly leadto the possibility of therapy and expansion of the choices.

Regarding Embodiment of Molecular Target Drugs:

As the embodiment of molecular target drugs, various substances havebeen studied and developed such as a therapeutic antibody, a lowmolecular weight drug, a peptide drug, a biological protein preparationsuch as cytokine, a siRNA, aptamer and the like. When an antibody isused as a therapeutic agent, due to its specificity, it is useful intreating pathological conditions in which a tumor specific antigenexhibits a property of different cells. The antibody binds to a tumorspecific antigen which is a protein expressing on the cell surface andeffectively acts upon such cells. The antibody has a characteristic oflong blood half life and high specificity for its antigen and is alsoparticularly useful as an antitumor agent. For example, when an antibodytargets at a tumor-specific antigen, it can be expected that theadministered antibody accumulates into the tumor and thereby attacks thetumor cell via complement-dependent cytotoxicity (CDC) andantibody-dependent cellular cytotoxicity (ADCC) based on the immunesystem. In addition, by binding a radioactive substance, a cytotoxicsubstance and the like to an antibody, it becomes possible to transferan agent efficiently to the tumor part and thereby to allow to actthereon. At the same time, since it can decrease the amount of thereached agent to non-specific other tissues, reduction of side effectscan also be expected. Termination or regression of growth of tumor canbe expected by activity of antibody accumulated at tumor selectively.Administering antibody will be able to select from antibodies, havingagonistic activity when a tumor-specific antigen has an activity toinduce cell death, or having neutralization activity when atumor-specific antigen relates to in the growth and survival of cells.Due to the above characteristics, it is considered that antibodies aresuited in applying as antitumor agents.

Regarding Therapeutic Antibodies:

In the original antibody preparation, a mouse was used as the animal tobe immunized. However, use of mouse antibodies as drugs in vivo islimited due to a large number of reasons. A mouse antibody which can berecognized as a foreign substance in a human body can induce so-called“human anti-mouse antibody” namely “HAMA” response (Non-patent Reference8). Further, the Fc region of mouse antibody is not effective forstimulation of human complement or cellular cytotoxicity.

As one of the approaches for avoiding such problems, a chimeric antibodyhas been developed (EP patent application No. 120694, EP patentapplication No. 125023). The chimeric antibody contains parts ofantibodies derived from two or more species (mouse antibody variableregion, human antibody constant region and the like). An advantageouspoint of such a chimeric antibody is that it keeps the characteristicsof a mouse antibody but can stimulate human complement or cellularcytotoxicity since it has human Fc. However, it is known that such achimeric antibody also induces “human anti-chimeric antibody” namely“HACA” response (Non-patent Reference 9).

Further, it has been developed a recombinant antibody in which onlyparts of an antibody, complementarity determining regions (“CDR”), weresubstituted (Patent References 1 and 2). By the use of a CDR graftingtechnique, an antibody comprising mouse CDR and human variable regionframework and constant region, so-called “humanized antibody”(Non-patent Reference 10).

Regarding TIM-3:

TIM gene family comprises eight genes in mice and three genes in humans,and these genes are located at chromosome 11 and at gene region 5q33,respectively (Non-patent Document 11). These gene regions are known torelate to autoimmune diseases and allergic diseases. TIM protein is atype I transmembrane protein having a structurally conservedimmunoglobulin variable (IgV) domain and a mucin domain. TIM protein wasconsidered to be specifically expressed on T cells and directly regulatethe T cell activity, but there are recent reports on expression of TIM-3protein in antigen-presenting cells and on their functions (Non-patentDocument 12). According to the crystal structure analysis, the TIMprotein has a conserved protein structure and has a ligand binding sitein the IgV domain.

TIM-3 was identified as a molecule specifically expressed on mouse Th1cells but not on Th2 cells (Non-patent Document 13). In mice, by bindingof TIM-3 to its ligand, galectin9, apoptosis is induced in a mouse Th1cell, the Th1 response is inhibited, and then to lead to induction ofperipheral tolerance. In humans, as similar to mice, TIM-3 isselectively expressed on Th1-cells, as well as phagocytic cells such asmacrophages and dendritic cells. It is found that the reduction ofexpression of human TIM-3 by siRNA or inhibition by a blocking antibodyincreased the secretion of interferon γ (IFN-γ) from CD4 positiveT-cells. This supports the inhibitory role of TIM-3 in human T cells.Analysis of clinical samples from autoimmune disease patients showed noexpression of TIM-3 in CD4 positive cells. In particular, expressionlevel of TIM-3 is lower and secretion of IFN-γ is higher in T cellclones derived from the cerebrospinal fluid of patients with multiplesclerosis than those in clones derived from normal healthy persons(Non-patent Document 14).

There are reports on relation of TIM-3 with allergic diseases and/orasthma (Patent Documents 3 and 4). However, the relation between TIM-3and blood cancer is disclosed only in the report on the microarrayanalysis of hematopoietic stem cell from acute myeloid leukemia patientsand normal hematopoietic stem cells (Non-patent Document 15) and many ofthe relation between TIM-3 and blood cancer has not been found out.

CITATION LIST Patent Document

-   Patent Document 1: GB Patent Application No. GB2188638A-   Patent Document 2: U.S. Pat. No. 5,585,089-   Patent Document 3: WO96/27603-   Patent Document 4: WO2003/063792

Non-Patent Document

-   Non-patent Document 1: Osawa M et al., Science. 273:242-5. (1996)-   Non-patent Document 2: Bonnet D and Dick J E, Nat Med. 3:730-7    (1997)-   Non-patent Document 3: Goodell M A et al., J Exp Med. 183:1797-806    (1996)-   Non-patent Document 4: Yamazaki S et al., EMBO J. 25:3515-23 (2006)-   Non-patent Document 5: Ishikawa F et al., Nat Biotechnol.    25:1315-21. (2007)-   Non-patent Document 6: Bao S et al., Nature. 444:756-60 (2006)-   Non-patent Document 7: Bernstein I D, Leukemia (2000) 14, 474-475-   Non-patent Document 8: Schiffetal., Canc. Res. (1985), 45, 879-885-   Non-patent Document 9: Bruggemann, et al., J. Exp. Med., 170,    2153-2157, 1989-   Non-patent Document 10: Riechmann, et al., Nature (1988), 332,    323-327-   Non-patent Document 11: Hafler D A et al., J Exp Med. 205:2699-701    (2008)-   Non-patent Document 12: Anderson A C et al., Science 318:1141-3    (2007)-   Non-patent Document 13: Monney L et al., Nature 415: 536-41. (2002)-   Non-patent Document 14: Koguchi K et al., J Exp Med. 203:1413-8.    (2006)-   Non-patent Document 15: Majeti R et al., Proc Natl Acad Sci USA.    2009 Feb. 13. PMID: 19218430

SUMMARY OF THE INVENTION Technical Problems

As a problem of a conventional treating method for malignant tumor, thefollowing three problems can be mentioned: firstly, side effects affectthe entire body due to an antitumor agent, radiation therapy and thelike; secondly, the possibility of relapse of the tumor increases due toremaining rare cancer stem cells which exhibit resistance to treatment;and thirdly, there are a few molecular target drug among drugs in thefield of blood tumor.

The first object of the present invention is to provide an agent forprevention, diagnosis, or treatment of various blood tumors bydeveloping an antitumor substance which can bind selectively and attacka malignant tumor cell in which TIM-3 is expressed.

The second object of the present invention is to provide use of TIM-3molecule based on the finding that TIM-3 is expressed in a blood tumorcell.

As described above, there is high expectation for a novel moleculartarget drug for a blood tumor including leukemia and it is consideredthat the finding of a novel target molecule and use thereof lead to adevelopment of safe and effective treating methods. Since an antibodyoriginally has recognition specificity, it is useful as a mean formolecular targeting. Therefore, the present inventors made an earneststudy on expression of human TIM-3 in blood tumors and preparation of anantibody to human TIM-3 and, as a result, they found use of TIM-3 as atarget for treatment.

Solution to Problems

As solutions to the above problem, for the first problem, the presentinvention provides to use as an agent for treatment targeting a moleculewhich is highly expressed in a cancer cell as compared to a normal cell.By using such a molecule, the treatment in which drug efficacy on cancercells becomes large and adverse effects on normal cells becomes small ispossible. Besides, the second problem can be solved by using a moleculewhich is expressed in a cancer stem cell as a target. The third problemcan be solved by an agent for treatment which meets above two pointssince the number of options of treating methods using a molecular targetdrug increases.

The present invention provides a novel molecular target drug fortreating a blood tumor by using a human monoclonal antibody to TIM-3which is a highly expressed gene in a malignant tumor and therebyattacking a malignant tumor cell including a cancer stem cell.

The present invention can be summarized as follows.

-   (1) A method for treatment, comprising administering an anti-TIM-3    antibody to a subject who is suspected to have blood tumor in which    a cell expressing TIM-3 is recognized in bone marrow or peripheral    blood or a subject who has received treatment for blood tumor in    which a cell expressing TIM-3 is recognized in bone marrow or    peripheral blood.-   (2) A composition for preventing or treating blood tumor of a    subject in which a cell expressing TIM-3 is recognized in bone    marrow or peripheral blood, comprising an anti-TIM-3 antibody as an    active ingredient.-   (3) A composition for detecting blood tumor in which a cell    expressing TIM-3 is recognized in a cell in bone marrow or    peripheral blood from a biological sample derived from a subject,    comprising an anti-TIM-3 antibody.-   (4) The method or composition according to any one of (1) to (3),    wherein the cell is a cell fraction selected from the following (a)    to (c):

(a) Lin(−)CD34(+)CD38(−)

(b) Lin(−)CD34(+)CD38(+)

(c) Lin(−)CD34(−)

-   (5) The method or composition according to any one of (1) to (4),    wherein the blood tumor is AML.-   (6) The method or composition according to any one of (1) to (4),    wherein the blood tumor is lymphoma, MDS or CML.-   (7) The method or composition according to any one of (1) to (6),    wherein the blood tumor is recurrence and/or refractory.-   (8) The method or composition according to any one of (1) to (7),    wherein the anti-TIM-3 antibody is an anti-TIM-3 monoclonal    antibody.-   (9) The method or composition according to (8), wherein the    anti-TIM-3 monoclonal antibody is an antibody having ADCC activity    and/or CDC activity.

Advantageous Effect of the Invention

TIM-3 is known as a gene which controls immune function in a livingbody. In the present invention, the inventors apply the newly foundnature of TIM-3, that is, express in blood tumor, to establish an usageof an agent for treatment or diagnosis and further the gene as a targetfor isolating a tumor cell by using an antitumor substances (preferablyanti-TIM-3 antibody) targeting TIM-3 expressing cells.

Since now, some molecular target drugs have been developed in the fieldof blood tumor. However, a few of them target a cancer stem cell. Forexample, AML treating drug, Mylotarg, which targets a CD33 protein isone of them (Bernstein, Leukemia (2000) 14, 474-475). However, asdescribed above, it has limitations such as expression level of CD33,recurrence, age, resistance to chemical therapy.

In Examples of the present invention, cytotoxicity of the anti-TIM-3antibody on TIM3 expressing cells or AML primary cells is found. Thisactivity suggests therapeutic effects on various blood diseases by theanti-TIM-3 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M0) patient.

FIG. 2 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M1) patient.

FIG. 3 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M2) patient.

FIG. 4 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M3) patient.

FIG. 5 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M4) patient.

FIG. 6 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M5) patient.

FIG. 7 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an AML (M6) patient.

FIG. 8 shows a result of cytotoxic assay for AML primary cells using ananti-TIM-3 polyclonal antibody.

FIG. 9 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an MDS patient.

FIG. 10 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from a CML patient.

FIG. 11 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells derived from an ALL patient.

FIG. 12 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow Lin(−)CD34(+)CD38(−)cells and Lin(−) CD34(+)CD38(+) cells derived from an relapsed AMLpatient.

FIG. 13 shows a result of multicolor flow cytometric analysis ofexpression of human TIM-3 molecule on bone marrow and mobilizedperipheral blood Lin(−) CD34(+)CD38(−) cells and Lin(−)CD34(+)CD38(+)cells derived from a normal healthy donors.

FIG. 14 shows a result of flow cytometric analysis of expression ofhuman TIM-3 molecule on normal peripheral blood cells derived from anormal healthy person.

FIG. 15 shows a result of flow cytometric analysis of expression ofhuman TIM-3 molecule on human cell lines.

FIG. 16 shows a result of ADCC assay of AML cell line using anti-TIM-3polyclonal antibody. The ordinate shows specific lysis rate.

FIG. 17 shows a result of CDC assay of AML cell line using anti-TIM-3polyclonal antibody.

MODE FOR CARRYING OUT THE INVENTION

(Detailed Description of Specified Desirable Embodiments)

Headings of the sections to be used in this specification are only forthe purpose of organization and should not be interpreted as limitationto the main subject to be described. All of the cited references citedin this application are clearly incorporated by references into thisspecification for optional purposes.

(Outline)

This invention provides an agent for prevention, diagnosis or treatmentfor malignant tumor in which a TIM-3 expressing cell is a target.

In the specific embodiment, the antibody has a binding activity to aTIM-3 molecule and can induce reduction or removal of a TIM-3 expressingcell by an immune system mainly centered on effector cells.

The antibody relates to a functional control of TIM-3 expressing celland induces survival, growth, resting, cell death or the like.

The antibody relates to a functional control of TIM-3 expressing celland increases or decreases the amount of production of cytokines orinterferon secreted from the cells.

(TIM-3)

TIM gene family consists of eight genes in mouse and three genes inhuman, and they are located at chromosome 11 and at gene region 5q33.These gene regions are known to be related with autoimmune diseases andallergic diseases. TIM protein is a type I transmembrane protein havinga structurally conserved immunoglobulin variable (IgV) domain and amucin domain.

In addition, a known ligand of TIM-3, galectin-9, is reported to expresson peripheral leukocytes and lymphoid tissue in human (Wada J et al., JBiol Chem. 272: 6078-86 (1997)). It is known that human galectin-9 bindsto GLUT-2 transporter protein and various oligosaccharide other thanTIM-3 (Nagae M et al., J Mol Biol. 375: 119-35 (2008)).

In addition, TIM-3 is also called as HAVCR2. TIM-3 includes mammal (suchas primate and human) TIM-3. Therefore, the anti-TIM-3 antibody of thepresent invention includes an antibody which specifically binds to thesequence of mammal TIM-3 such as human TIM-3. The sequence of TIM-3 suchas human TIM-3 includes polymorphic variants. A non-specific example ofthe full length human TIM-3 includes the sequence described as follows:

(SEQ ID NO: 1) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPL GCRFAMP(Antibody)

The antibody is used in a most broad sense and includes a monoclonalantibody, a polyclonal antibody, a multivalent antibody, a multispecificantibody (e.g., bispecific antibody) and also antibody fragments as longas these exhibit the desired biological activity.

The antibody contains a mature heavy chain or light chain variableregion sequence. In addition, the antibody also includes a modified formand variant form such as substitutions within or outside of a constantregion, a complementary determining region (CDR) or a framework (FR)region of a mature heavy or light chain variable region sequence of theantibody, and the like. In a specific embodiment, the substitutionincludes a conservative amino acid substitution is included in thesubstitution.

In addition, the antibody also includes a subsequence of the matureheavy chain or light chain variable region sequence. In a specificembodiment, the subsequence is selected from Fab, Fab′, F(ab′)₂, Fv, Fd,single chain Fv (scFv), disulfide bond Fv (sdFv) and VL or VH.

In addition, the antibody also includes a heterogeneous domain. In aspecific embodiment, the heterogeneous domain includes a tag, adetectable label or a cytotoxic agent.

Examples of the antibody include a monoclonal antibody and a polyclonalantibody and any isotype or subclass thereof. In a specific embodiment,the aforementioned antibody is an isotype of IgG (e.g., IgG1, IgG2, IgG3or IgG4), IgA, IgM, IgE or IgD. The “monoclonal” antibody means anantibody that is based upon a single clone including a eukaryote clone,a prokaryote clone or a phage clone, obtained from a single cloneincluding a eukaryote clone, a prokaryote clone or a phage clone, orinduced from a single clone including a eukaryote clone, a prokaryoteclone or a phage clone. Accordingly, the “monoclonal” antibody is astructurally defined substance and not a method by which it is produced.

The TIM-3 antibody, anti-TIM-3 and anti-TIM-3 antibody mean an antibodywhich specifically binds to TIM-3. The specific binding means that it isselective for the epitome presenting in TIM-3. The specific binding canbe distinguished from non-specific binding using a known assay in thetechnical field (e.g., immunoprecipitation, ELISA, flow cytometry, andWestern blotting).

When all or a part of antigen epitopes to which an anti-TIM-3 antibodyspecifically binds are present in different proteins, there is apossibility that this antibody can bind to the different proteins.Therefore, there is a possibility that the TIM-3 antibody specificallybinds to other protein having high sequence or structural homology toTIM-3 epitope depending on the sequence or structural homology of TIM-3epitope. Accordingly, there is a possibility that TIM-3 antibody bindsto a different protein when an epitope having sufficient sequence orstructural homology is present in the different protein.

The TIM-3 antibody includes isolated and purified antibodies. Theantibody of the invention including an isolated or purified TIM-3antibody includes human.

The term “(be) isolated” to be used as a modifier of a composition meansthat the composition is prepared by the hand of man or separated fromone or more other components in in vivo environment presenting in naturegenerally by one or more manipulative steps or processes. In general, acomposition separated in this manner does not substantially contain oneor more materials with which they normally associate in nature, such asone or more proteins, nucleic acids, lipids, carbohydrates and cellmembranes. Because of this, the isolated composition is separated fromother biological components in the cells of the organism in which thecomposition naturally occurs, or from the artificial medium in which itis produced (e.g., by synthesis or cell culture). For example, anisolated TIM-3 antibody can be obtained from an animal in which theantibody is produced (e.g., non-transgenic mammals or transgenic mammals(such as rodents (mouse), the ungulates (cattle) and the like)) and isseparated from other polypeptides and nucleic acids. Accordingly, it isconsidered that the serum containing an antibody obtained from such ananimal is isolated. The term “(be) isolated” does not excludealternative physical forms, and for example, an isolated antibody couldinclude antibody subsequences, chimerized, multimerized or derivatizedforms.

The term “(be) purified” to be used as a modifier of a compositionrefers to a composition which is free of most of or substantially all ofthe materials with which it typically associates in nature. In general,a purified antibody is obtained from the components generally presentingin the antibody environment. Because of this, it is considered that anantibody supernatant which is separated from a cell culture mixture ofan antibody producing hybridoma is purified. Accordingly, the “(be)purified” does not require absolute purity and is context specific.Furthermore, the “(be) purified” composition can be combined with one ormore other molecules. Because of this, the term “(be) purified” does notexclude combination of composition. The purity can be determined by anoptional appropriate method such as UV spectrometry, chromatography(e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or Coomassiestaining), sequence analysis (peptide and nucleic acid) and the like.

The “(be) purified” protein and nucleic acid include a protein and anucleic acid which are obtained by a standard purification method. Also,a protein and a nucleic acid obtained by recombination expression in ahost cell and chemical synthesis are also included in this term. Inaddition, the “(be) purified” can also refer to a composition in whichthe level of contaminants is lower than the level which is acceptable toa regulatory agency for administration to human or non-human animals,such as the Food and Drug Administration (FDA).

The anti-TIM-3 antibody also includes one which can specifically bind toTIM-3 expressed on the cell. In specific embodiment, the anti-TIM-3antibody can specifically bind to TIM-3 expressing blood tumor cells(AML cells, CML cells, myelodysplastic syndromes (MDS) cells, ALL cells,CLL cells, Multiple Myeloma cells, or various lymphoma such as B-celllymphoma, T cell lymphoma, NK-cell lymphoma), helper T cell (e.g., Th1cell, Th17 cell), an antigen-presenting cells (such as dendritic cells,monocytes, macrophage and subtypes thereof (e.g., hepatic stellate cell,osteoclast, microglia, intraepidermal macrophage, dust cell (alveolarphagocyte))) or TIM-3 expressed in a TIM-3 gene transfected cell.

Examples of the anti-TIM-3 antibody include an antibody whichspecifically binds to an epitope in the amino acid sequence ofextracellular region of TIM-3. In the specific embodiments, exemplaryanti-TIM-3 antibody specifically binds to three epitopes on TIM-3 asdecided by across-blocking assay. A non specific exemplary sequence ofthe human TIM-3 extracellular region is as follows.

(SEQ ID NO: 2) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSG ATIR

Examples of the anti-TIM-3 antibody include an antibody which can bindto TIM-3 existing on one or more kinds of the cells in vivo, isolatedprimary cultured cells, subcultured cells, cultured cells andimmortalized cells. Specific examples of non-specific cell types whichcan express TIM-3 include AML cells, other blood tumor cells (such asCML cells, ALL cells, CLL cells, MDS cells, Multiple Myeloma cells,various lymphoma cells (e.g., B-cell lymphomas, T cell lymphoma, NK celllymphoma and the like)) and non-blood tumor cells. Examples of non-bloodtumor cells include monocytes, dendritic cells, macrophages, helper Tcells, natural killer cells, myeloid progenitor cells and lymphoidprogenitor cells. TIM-3 can be expressed in a cell which originally doesnot express TIM-3 by transfection or transformation using the nucleicacid encoding TIM-3. The anti-TIM-3 antibody which can bind TIM-3 canbind one or more transfectant or transformant expressing or producingTIM-3.

In the present specification, AML cells, CML cells, ALL cells, CLLcells, MDS cells, Multiple Myeloma cells, various lymphoma cells, suchas B-cell lymphomas, T cell lymphoma, NK cell lymphoma, include theircancer stem cells.

The cancer stem cell is one of the population of cell constituting tumorand it is represented by Lineage(−)CD34(+)CD38(−) myeloid cell. Asanother name corresponding to disease, a tumor stem cell, a leukemiastem cell and the like are known.

Among the blood tumor cells, leukemia cells obtained from bone marrow ofa patient of leukemia (such as leukemia stem cells and blast cells) canbe fractionated according to a differentiation antigen (Lineage marker)and a cell surface marker such as CD34 and CD38. Most of leukemia stemcells reside in the Lineage(−)CD34(+)CD38(−) cell fraction. On the otherhand, the blast cell is identified morphologically. Although it is aLineage(−)CD34(+)CD38(+) cell, it also exists in various cell groupsincluding Lineage(−)CD34(+)CD38(−) cell fraction andLineage(−)CD34(−)cell fraction.

The TIM-3 antibody includes an antibody which binds to TIM-3 andmodulates function or activity of TIM-3 in vivo or in vitro (e.g., in asubject). In the specification, the “to modulate” and the grammaticalvariations thereof when used in relation to the activity or function ofTIM-3 mean that the TIM-3 activity or function is detectably affected,modified or altered. Accordingly, the TIM-3 antibody which modulates theactivity or function of TIM-3 is an antibody that provides influence,modification or alteration such that one or more of the TIM-3 activityor function can be detected, and such an activity or function of TIM-3can includes, for example, binding of TIM-3 with an TIM-3 ligand, anTIM-3-mediated signal transduction or an TIM-3-mediated cell response ora cell response that can be modulated by TIM-3, or the activity orfunction of other TIM-3 described in the specification or, otherwise, iscommonly known or can be known.

Examples of various non-limited TIM-3 activities and functions which canbe modulated include TIM-3 mediated signal transduction or TIM-3mediated cellular response, cellular response which can be modulated viaTIM-3, cell proliferation or cell expansion (e.g., AML cells, CML cells,ALL cells, CLL cells, MDS cells, Multiple Myeloma cells, variouslymphoma cells, such as B-cell lymphomas, T cell lymphoma, and NK celllymphoma, monocytes, dendritic cells, macrophages, helper T cells,natural killer cells, myeloid progenitor cells and lymphoid progenitorcells), cell survival or cell death such as apoptosis (e.g., AML cells,CML cells, ALL cells, CLL cells, MDS cells, Multiple Myeloma cells,various lymphoma cells, such as B-cell lymphomas, T cell lymphoma, andNK cell lymphoma, monocytes, macrophages, helper T cells, natural killercells, myeloid progenitor cells and lymphoid progenitor cells),cytokines (e.g., Th1, Th2 and non-Th1/Th2 cytokines) and expression orproduction of interferon, expression or production of anti-apoptosisprotein or proapoptosis protein, treatment, suppression or improvementof disorder, disease, physiological condition, pathological conditionand symptom of them. Specific cytokines to be modulated are not limitedand examples include IL-1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-14,IL-16, IL-17, IL-23, IL-26, TNF-α, and interferon γ (in vitro or invivo). Specific anti-apoptosis proteins and proapoptosis proteins arenot limited and examples include Bcl-xL, Bcl-2, Bad, Bim, and Mcl-1.

Therefore, examples of anti-TIM-3 antibody described in the presentspecification include an antibody which modulates TIM-3 mediated one ormore signal transduction or TIM-3 mediated cellular response, cellularresponse which can be induced by TIM-3, cell proliferation (e.g., AMLcells, CML cells, ALL cells, CLL cells, MDS cells, Multiple Myelomacells, various lymphoma cells such as B-cell lymphomas, T cell lymphoma,and NK cell lymphoma, monocytes, macrophages, helper T cells, naturalkiller cells, myeloid progenitor cells and lymphoid progenitor cells),cell survival or apoptosis (e.g., AML cells, CML cells, ALL cells, CLLcells, MDS cells, Multiple Myeloma cells, various lymphoma cells such asB-cell lymphomas, T cell lymphoma, and NK cell lymphoma, monocytes,macrophages, helper T cells, natural killer cells, myeloid progenitorcells and lymphoid progenitor cells), expression or production ofcytokines (e.g., Th1, Th2 and non-Th1/Th2 cytokines, IL-17, IL-23 andIL-26) and interferon (e.g., Th1, Th2, non-Th1/Th2, IL-1, IL-2, IL-4,IL-5, IL-6, IL-9, IL-10, IL-14, IL-16, IL-17, IL-23, IL-26, TNF-α,interferon γ, and GM-CSF (in vivo or in vitro) and the like), expressionof anti-apoptosis protein or proapoptosis protein (e.g., Bcl-xL, Bcl-2,Bad, Bim and Mcl-1), treatment, suppression or improvement of disorder,disease, physiological condition, pathological condition and symptom. Inthe specific embodiments, anti-TIM-3 antibody of the present inventioncan modulate expansion or survival of AML cell, number of other bloodtumor cell (e.g., CML cells, ALL cells, CLL cells, MDS cells, MultipleMyeloma cells, or various lymphoma cells such as B-cell lymphomas, Tcell lymphoma, and NK cell lymphoma), growth or survival of non-bloodtumor cell such as monocytes, dendritic cells, macrophages, helper Tcells, natural killer cells, myeloid progenitor cells and lymphoidprogenitor cells, and reduces, disappears or depletes AML cells, CMLcells, ALL cells, CLL cells, MDS cells, Multiple Myeloma cells, andvarious lymphoma cells, such as B-cell lymphomas, T cell lymphoma, andNK cell lymphoma.

The TIM-3 antibody includes a modified form such as a substitutionproduct (e.g., an amino acid substitution product) which is also calledas “variant”, an addition product, deletion product (e.g., a subsequenceor fragment) and the like. Such modified antibody forms and variantsretain at least partial function or activity of the TIM-3 antibody shownby the invention, such as binding with TIM-3, or modulation of activityor function (e.g., TIM-3 signal transduction) of TIM-3. Accordingly, themodified TIM-3 antibody can retain the ability to modulate, for example,at least partial of TIM-3 binding or one or more of the TIM-3 functionsor activities (e.g., signal transduction, cell response and the like).

According to this specification, the term “to alter” (“to modify”) andthe grammatical variations thereof means that the compositionderivarates a reference composition. The altered proteins, nucleic acidsand other compositions can have higher or lower activities than areference unmodified protein, nucleic acid or other composition or canhave a different function from a reference unmodified protein, nucleicacid or other composition.

Such an antibody containing an amino acid substitution can be encoded bynucleic acid. Accordingly, the present invention also provides anucleotide sequence encoding an antibody containing an amino acidsubstitution.

The term “identity” or “identical” means that two or more referencedsubstances are the same. Accordingly, when two protein sequences (e.g.,TIM-3 antibodies) are identical, they have the same amino acid sequencesat least within the referenced regions or portion. The term “identicalregion” means an identical region of two or more referenced substances.Thus, when two protein sequences are identical over one or more sequenceregions, they have identity within the regions. “Substantial identity”means that a molecule is structurally or functionally conserved suchthat the molecule has or is predicted to have at least partial functionor activity of one or more of reference molecule functions or activitiesor relevant/corresponding region or a portion of the reference moleculeto which it shares identity. Thus, polypeptides having substantialidentity (e.g., TIM-3 antibodies) have or are predicted to have at leasta part of the activity or function as a referenced polypeptide (e.g.,TIM-3 antibody). For example, in a specific embodiment, it is consideredthat a TIM-3 antibody having one or more modifications (e.g., amino acidsubstitution, deletion or addition) which retain at least partialactivity or function of the unmodified TIM-3 antibody has substantialidentity to the reference TIM-3 antibody.

Due to variations between structurally related protein and functionallyrelated protein, the amount of sequence identity may vary with what isrequired to retain functions of the protein, activity of the protein,function of the region, or activity of the region. In the case ofprotein, an activity or function can be retained by the presence ofmerely 30% of amino acid sequence identity, but in general, higheridentity of 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97% or 98%, to thereference sequence is present. The extent of identity between twosequences can be confirmed using a computer program or mathematicalgorithm conventionally known in the technical field. In such analgorithm which calculates ratio of sequence identity (homology), ingeneral, sequence gaps and mismatches over the comparison region areaccounted. For example, BLAST (e.g., BLAST 2.0) retrieval algorithm(e.g., see Altschul et al., J. Mol. Biol., 215: 403 (1990), publiclyavailable through NCBI) has the following illustrative retrievalparameters: mismatch −2; gap start 5; gap elongation 2. In thepolypeptide sequence comparison, the BLASTP algorithm is typically usedin combination with a scoring matrix such as PAM 100, PAM 250, BLOSUM62, BLOSUM50.FASTA (e.g., FASTA 2 and FASTA 3) and the like, and SSEARCHsequence comparison program is also used for determining the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444 (1988);Pearson, Methods Mol. Bio., 132: 185 (2000); and Smith et al., J. Mol.Biol., 147: 195 (1981)). A program has also been developed fordetermining protein structural similarity using topological mappingbased on Delaunary (Bostick et al., Biochem. Biophys. Res. Commun., 304:320 (2003)).

A “conservative substitution” is a substitution of one amino acid by abiologically, chemically or structurally similar residue. Biologicalsimilarity means that a biological activity such as TIM-3 bindingactivity is not destroyed by the substitution. Structural similaritymeans that amino acids have side chain with similar length (e.g.,alanine, glycine and serine) or have similar size. Chemical similaritymeans that the residues have the same charge or are both hydrophilic orhydrophobic. Specific examples include substitution of one hydrophobicresidue such as isoleucine, valine, leucine, and methionine with otherresidue, or the substitution of one polar residue with other residuesuch as the substitution of arginine with lysine, the substitution ofglutamic acid with aspartic acid, or the substitution of glutamine withasparagine, and the substitution of serine with threonine.

In addition, examples of the modified antibody include peptide mimeticshaving one or more D-amino acids substituted with L-amino acids (and amixture thereof), structural and functional analogs such as synthesizedor non-natural amino acids or amino acid analogs, and derivatized formthereof. Examples of modification include a cyclic structure such as anend-to-end amide bond between the amino and carboxy-terminus of themolecule or intra- or inter-molecular disulfide bond.

Additional specific non-limiting examples of the amino acidmodifications include partial sequence (subsequence) and fragment ofTIM-3. Exemplary subsequence and fragment of TIM-3 include a part of theTIM-3 sequence to which the exemplary TIM-3 antibody of the inventionbinds. Also, the exemplary subsequence and fragment of TIM-3 include animmunogenicity region such as a part of the TIM-3 to which the exemplaryTIM-3 antibody of the invention binds.

The TIM-3 antibody subsequence and fragment can have a binding affinityas the full length antibody, a binding specificity as the full lengthantibody or one or more activities or functions as the full lengthantibody, such as the function or activity of an TIM-3 antagonist oragonist antibody. The terms “functional subsequence” and “functionalfragment” in the case of referring to the antibody mean an antibodyportion which retains one or more functions or activities as the fulllength reference antibody, such as at least a part of the function oractivity of TIM-3 antibody. For example, an antibody subsequence whichbinds to TIM-3 or a fragment of TIM-3 is considered a functionalsubsequence.

The antibody subsequence and fragment can be combined. For example, a VLor VH subsequence can be connected by a linker sequence and thereby canform a VL-VH chimeric body. A combination of single chain Fv (scFv)subsequences can be connected by a linker sequence and thereby can forma scFv-scFv-chimeric body. The TIM-3 antibody subsequence and fragmentinclude a single chain antibody or variable region alone or incombination with all or a portion of other TIM-3 antibody subsequence.

The antibody subsequence and fragment can be prepared by hydrolysis ofthe antibody by its proteolysis for example by a pepsin or papaindigestion of the whole antibody. The antibody subsequence and fragmentobtained by enzymatic cleavage with pepsin provide a 5S fragmentrepresented by F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to form a 3.5S Fab′ monovalent fragment.Alternatively, an enzymatic cleavage using pepsin directly produces twomonovalent Fab′ fragments and Fc fragment (see e.g., U.S. Pat. Nos.4,036,945 and 4,331,647; and Edelman et al., Methods Enzymol., 1: 422(1967)). Other methods of cleaving an antibody, such as separation ofheavy chain for forming a monovalent light chain-heavy chain fragment,further cleavage of the fragment or other enzymatic or chemical methodmay be used.

A protein and an antibody, as well as subsequence thereof and fragmentcan be prepared using a genetic method. The technology includes the fullor partial gene encoding a protein or an antibody is expressed in a hostcell such as a COS cell and E. Coli. A recombinant host cell synthesizesthe full or subsequence such as scFv (such as Whitlow et al, In:Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird et al,Science 242:423 (1988); and U.S. Pat. No. 4,946,778). A single chain Fvand an antibody can be prepared in accordance with the procedure asdescribed in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al,Methods Enzymol 203:46 (1991); Shu et al, Proc. Natl. Acad. Sci. USA90:7995 (1993); and Skerra et al, Science 240:1038 (1988).

The modified form includes a derivatized sequence such as amino acids inwhich the free amino groups form amine hydrochloride, p-toluenesulfonylgroup and carbobenzoxy group; the free carboxy groups which form a saltor methyl and ethyl ester; and the free hydroxyl groups form O-acyl orO-alkyl derivatives, and naturally existing amino acid derivatives suchas 4-hydroxyproline (derivative of proline), 5-hydroxylysine (derivativeof lysine), homoserine (derivative of serine), ornithine (derivative oflysine) and the like. The modification can be carried out using a methodconventionally known in the technical field (e.g., site-specificdeletion or insertion mutagenesis based on PCR, chemical modificationand mutagenesis, crosslinking and the like).

Addition products and insertion products are included in the modifiedforms of protein (e.g., antibody), nucleic acid and other compositions.For example, the addition can be a covalent or non-covalent bond withany type of molecules of protein (e.g., antibody), nucleic acid or othercompositions. In general, addition and insertion confer differentfunction or activity.

Fusion (chimeric) polypeptides or nucleic acid sequences are included inthe addition product and insertion product, and these are sequenceshaving one or more molecules which are generally not present in thereference native (wild type) sequence covalently attached to theaforementioned sequence. A specific example is an amino acid sequence ofother protein (e.g., an antibody) for producing a multifunctionalprotein (e.g., a multispecific antibody).

Also, the antibody of the invention include a chimeric or fusion productin which one or more additional domains are covalently linked thereto inorder to impair a different or complementary function or activity.Examples of the antibody include a chimeric or fusion product which doesnot naturally present in natural and in which two or more amino acidsequences are mutually bonded.

A linker sequence may be inserted between the protein (e.g., anantibody), nucleic acid or other composition and the addition product orinsertion product (e.g., a heterologous domain) so that the twosubstances maintain at least a part of different function or activity.The linker sequence may have one or more properties which can accelerateeither of the domains or can carry out mutual reaction with either ofthe domains, and such characteristics include impossibility to form aflexible structure and an ordered secondary structure or hydrophobicproperty or charging property. Examples of the amino acids which aregenerally found in the flexible protein regions include glycine,asparagine and serine. Other amino acids close to neutral such asthreonine and alanine may also be used in the linker sequence. Thelength of the linker sequence can be varied (e.g., see U.S. Pat. No.6,087,329). The linker further include chemical crosslinking agents andbinding agents (conjugating agents) such as a sulfo-succinimidylderivative (sulfo-SMCG, sulfo-SMPB), disuccinimidyl suberate (DSS),disuccinimidyl glutarate (DSG) and disuccinimidyl tartarate (DST).

Further examples of the addition include any one of glycosylation, fattyacid, lipid, acetylation, phosphorylation, amidation, formylation,ubiquitination and derivatiation by a protecting or blocking group and alarge number of chemical modifications. Other substitutions andpossibilities can be easily understood by those skilled in the art andare considered to be within the scope of the invention.

Such a modified sequence can be prepared using recombinant DNAtechniques which mediate cell expression or in vitro translation.Polypeptides and nucleic acid sequences can also be prepared by aconventionally known method in the technical field such as chemicalsynthesis using an automatic peptide synthesizer (see e.g., AppliedBiosystems, Foster City Calif.).

Modified and variant antibodies such as substitution products,subsequences addition products and the like can maintain detectableactivity of TIM-3 antibody. In an embodiment, the modified antibody hasthe activity to bind to TIM-3 molecule and induces reduction orelimination of TIM-3 expression cells by an immune system mainlycentering on an effector cell. The modified antibody relates to thefunctional control of TIM-3 expression cells and induces survival,growth, resting, cell death and the like of the cells. The cell deathincludes apoptosis, necrosis, autophagy and the like.

According to the invention, there are further provided a cell-freemethod (e.g., in a solution or by a solid phase) and a cell-based method(e.g., in vivo or in vitro) which screen, detect and identify TIM-3.These methods can be carried out in a solution in vitro using abiomaterial or sample, and in vivo for example using a sample of ananimal-derived cell (e.g., lymphocyte). In an embodiment, the methodcomprises a step of contacting a biomaterial or sample with aTIM-3-binding antibody under a condition of allowing binding of theantibody to TIM-3 and a step of assaying for binding of the antibody toTIM-3. The presence of TIM-3 is detected by binding of the antibody tobind to TIM-3. In an embodiment, TIM-3 is present in a cell or tissue.In another embodiment, the aforementioned biomaterial or sample isobtained from a mammal analyte.

The term “contacting” when it is used in relation to the compositionsuch a protein (e.g., TIM-3 antibody), a material, a sample or treatmentmeans a direct or indirect interaction between the composition (e.g.,TIM-3 antibody) and other referenced substance. Specific examples of thedirect interaction include bonding. Examples of specific examples of theindirect interaction include a case in which the composition acts uponan intermediate molecule and this intermediate molecule then acts uponthe referenced substance. Accordingly, for example, contacting a cell(e.g., lymphocyte) to TIM-3 antibody includes the antibody to bind tothe cell (e.g., through binding to TIM-3) or to allow the antibody toact on an intermediate substance, followed by the action of thisintermediate substance upon the cell.

The terms “assaying” and “measuring” and grammatical variations thereofare synonymously used in the specification and mean either ofqualitative measurement and quantitative measurement or both ofqualitative measurement and quantitative measurement. When these termsare used in relation to binding, they include any means of evaluatingrelative amount, affinity or specificity of binding including variousmethods which are described in the specification and conventionallyknown in the technical field. For example, binding of the TIM-3 antibodywith TIM-3 can be assayed or measured by a flow cytometry assay.

(Preparation of Antibody)

The invention also provides a method for preparing a human TIM-3antibody having TIM-3-positive cytotoxicity. In an embodiment, themethod comprises administering a human TIM-3 extracellular regionconjugated with a human Fc recombinant protein or an TIM-3 geneintroduced cell to animals capable of expressing human immunoglobulin(e.g., transgenic mice or transgenic cattle); screening the animal forexpression of a anti-human TIM-3 antibody; selecting an animal producingthe anti-human TIM-3 antibody; isolating the antibody from the selectedanimal and deciding whether or not anti-human TIM-3 antibody has TIM-3antagonistic activity.

The TIM-3 protein suitable for the antibody preparation can be producedby any one of various standard protein purification and recombinantexpression techniques. For example, the TIM-3 sequence can be preparedby standard peptide synthesis techniques such as a solid phasesynthesis. In order to facilitate purification of the expressed orsynthesized protein, a portion of the protein may contain an amino acidsequence such as a FLAG tag, a T7 tag, a polyhistidine sequence or thelike. The protein is expressed inside the cells and can be purified. Theprotein can be expressed by a recombination method as a part of afurther large protein (e.g., a fusion or chimeric product). Theembodiment of the TIM-3 suitable for generating immune response includesTIM-3 subsequences such as an immunogenicity fragment. Furtherembodiment of TIM-3 includes a TIM-3 expressing cell, a TIM-3 containingpreparation or cell extract or fraction and a partially purified TIM-3.

The method for preparing polyclonal antibody and monoclonal antibody isconventionally known in the technical field. For example, TIM-3 or itsimmunogenicity fragment used for immunizing an animal by optionallyconjugating with a carrier such as keyhole limpet hemocyanin (KLH) orovalbumin (e.g., BSA) or mixing with an adjuvant such as complete orincomplete Freund's adjuvant. By isolating a spleen cell derived from animmunized animal which responds to TIM-3, it can be fused with myelomacell using hybridoma techniques. The monoclonal antibodies produced byhybridomas can be screened for reactivity with TIM-3 or immunogenicityfragment thereof.

The animal which can be immunized includes the primates, mouse, rat,rabbit, goat, sheep, cattle and guinea pig. The initial and anyoptionally subsequent immunization may be by intravenous route,intraperitoneal route, intramuscular route or subcutaneous route.Further, in order to increase immune response, the antigen can beconjugated with other protein such as ovalbumin, keyhole limpethemocyanin (KLH), thyroglobulin and tetanus toxoid, or can be mixed withan adjuvant such as complete Freund's adjuvant, incomplete Freund'sadjuvant and the like. The initial and any optionally subsequenceimmunization may be through intraperitoneal route, intramuscular route,intraocular route or subcutaneous route. The immunization may be at thesame concentration or different concentration of a TIM-3 preparation,and at regular or irregular intervals.

The animal includes those which are genetically modified to includehuman loci, and a human antibody can be prepared using the same.Examples of the transgenic animals with one or more human immunoglobulingenes, are described for example in U.S. Pat. No. 5,939,598, WO02/43478and WO02/092812. Using conventional hybridoma technique, a spleen cellswhich is isolated from immunized mouse having high responders to theantigen and fuse it with myeloma cell. A monoclonal antibody which bindsto TIM-3 can be obtained.

The method for producing a human polyclonal antibody and a humanmonoclonal antibody is further described (see, such as Kuroiwa et al,Nat. Biotechnol. 20: 889 (2002); WO98/24893; WO92/01047; WO96/34096;WO96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).

The term “human” when it is used in reference to an antibody means thatamino acid sequence of the antibody is completely the human amino acidsequence, namely is human heavy chain and human light chain variableregions and human constant region. Accordingly, all of the amino acidsare human amino acids or present in the human antibody. An antibodywhich is a non-human antibody can be made into a complete human antibodyby substituting the non-human amino acid residues with the amino acidresidues which are present in the human antibody. The amino acidresidues which are present in the human antibody, CDR region map andhuman antibody consensus residues are well known in the technical field(see e.g., Kabat, Sequences of Proteins of Immunological Interest, 4thedition, US Department of Health and Human Services, Public HealthService (1987); Chothia and Lesk (1987)). A consensus sequence of humanVH subgroup III based on the investigation carried out using 22 knownhuman VH III sequences as the object and a consensus sequence of humanVL κ chain subgroup I based on the investigation carried out using 30known human κ chain I sequences as the object are described in Padlan,Mol. Immunol., 31: 169 (1994) and Padlan, Mol. Immunol., 28: 489 (1991).Accordingly, the human antibody includes an antibody in which one ormore amino acid residues have been substituted with one or more aminoacids existing in an optional other human antibody.

Examples of the anti-TIM-3 antibody include antibodies prepared using aknown method in the technical field, such as CDR-grafting (EP 239,400;WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089),veneering or resurfacing (EP592,106; EP519,596; Padlan, MolecularImmunol. 28: 489 (1991); Studnicka et al., Protein Engineering 7: 805(1994); Roguska et al., Proc. Nat'l Acad. Sci. USA 91: 969 (1994)) andchain shuffling (U.S. Pat. No. 5,565,332). In order to produce ahumanized antibody, human consensus sequence (Padlan, Mol. Immunol.31:169 (1994); and Padlan, Mol. Immunol. 28: 489 (1991)) has been used(Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); and Prestaet al, J. Immunol. 151: 2623 (1993)).

The term “humanized” when it is used in relation to an antibody meansthat amino acid sequence of the antibody has one or more non-human aminoacid residues (e.g., mouse, rat, goat, rabbit and the like) ofcomplement determining region (CDR) which specifically binds to adesired antigen in an acceptor human immunoglobulin molecule and one ormore human amino acid residues (amino acid residues which are flankedwith CDR) in Fv framework region (FR). The antibody called “primatized”is within the scope of meaning of “humanized”, except that amino acidresidues of the acceptor human immunoglobulin molecule and frameworkregion can be any primate amino acid residues (e.g., monkey, gibbon,gorilla, chimpanzee, orangutan, macaque monkey) in addition to any humanresidues. Human FR residues of immunoglobulin can be substituted withcorresponding non-human residues. Accordingly, for example, in order toalter, generally to improve, antigen affinity or specificity, residuesin the CDR or human framework region can be substituted withcorresponding residues from the non-human CDR or framework region donorantibody. The humanized antibody can contain residues which cannot befound in the human antibody and donor CDR or framework sequence. Forexample, it can be predicted that FR substitution at a particularposition which cannot be found in human antibody or donor non-humanantibody can improve binding affinity or specific human antibody at thisposition. Antibody framework and CDR substitutions based on themolecular modeling are conventionally known in the technical field, forexample by the modeling of interaction of CDR and framework residues toidentify framework residues important for antigen binding and thesequence comparison for identifying unusual framework residues at thespecific position (see e.g., U.S. Pat. No. 5,585,089; and Riechmann etal., Nature, 332:323 (1988)).

Chimeric antibodies are included in the TIM-3 antibody. According tothis specification, the term “chimeric” and the grammatical variationsthereof when it is used in relation to antibodies mean that amino acidsequence of the antibody contains one or more portion which is derivedfrom two or more different species, is obtained or isolated from two ormore different species or is based on two or more different species. Forexample, a portion of the antibody can be human (e.g., constant region)and other portion of the antibody can be non-human (e.g., a mouse heavychain or a mouse light variable region). Accordingly, an example of thechimeric antibody includes an antibody in which the different portion ofthe antibody is derived from a different species. Different from thehumanized or primatized antibody, the chimeric antibody can have asequence of different species in an arbitrary region of the antibody.

The method for producing a chimeric antibody is known in the technicalfield (such as Morrison, Science 229: 1202 (1985); Oi et al.,BioTechniques 4: 214 (1986); Gillies et al., J. Immunol. Methods 125:191 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397). Forexample, in Munro, Nature 312: 597 (1984); Neuberger et al., Nature 312:604 (1984); Sharon et al., Nature 309: 364 (1984); Morrison et al.,Proc. Nat'l. Acad. Sci. USA 81: 6851 (1984); Boulianne et al., Nature312: 643 (1984); Capon et al, Nature 337: 525 (1989); and Traunecker etal., Nature 339: 68 (1989), a chimeric antibody in which a variableregion of antibody derived from one species is replaced by a variableregion of antibody derived from another species.

In addition, the anti-TIM-3 antibody can be prepared by hybridomatechnique, recombinant technique, and phage display technique, and acombination thereof (see U.S. Pat. Nos. 4,902,614, 4,543,439, and4,411,993; and also see Monoclonal Antibodies. Hybridomas: A NewDimensionin Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol et al, 1980, and Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, the second edition, 1988).

The human anti-human TIM-3 antibody of the invention was produced usingchromosome-transferred mice (KM mice (trademark)) immunized with variousforms of soluble form of recombinant human TIM-3 proteins or cellstrains expressing TIM-3 (WO02/43478, WO02/092812, and Ishida et al.,IBC's 11th Antibody Engineering Meeting, Abstract (2000)). The humananti-human antibody can not stain a non-transfected parent cell line butcan stain detectably a human TIM-3 stable transfected cell line, such asJurkat-TIM-3 cells and L929-TIM-3 cells.

The antibody of the invention can have κ light chain sequence or λ lightchain sequence, full length of either one of them as present innaturally existing antibody, a mixture thereof (namely a fusion productof κ chain sequence and λ chain sequence) and subsequences/fragmentsthereof. The naturally presenting antibody molecules contain two κ lightchains or two λ light chains.

In addition, the human TIM-3 antibody include an antibody whichspecifically binds to TIM-3 and does not inhibit or prevent the bindingof rat anti-human TIM-3 antibody 344823 (manufactured by R&D Systems,catalogue No. MAB2365 or FAB2365P).

In addition, the anti-human TIM-3 antibody includes an antibody whichspecifically binds to TIM-3 and inhibits binding of the other anti-humanTIM-3 antibody or does not inhibit binding of the other anti-human TIM-3antibody.

The method for producing an antibody which specifically binds to TIM-3can be provided as follows. In one embodiment, a method includesadministering a human TIM-3, subsequence or fragment (such asextracellular region of TIM-3) conjugated with human Fc recombinantprotein to an animal capable of expressing human immunoglobulin (e.g., atransgenic mice or transgenic cattle); screening the animal forexpression of human TIM-3 antibody; selecting an animal that produces ahuman TIM-3 antibody; isolating an antibody from the selected animal. Inone embodiment, using this method, one can determine whether theanti-human TIM-3 antibody has TIM-3 antagonist or agonist activity.

The invention further provides methods for producing human TIM-3antibodies that inhibits or prevents TIM-3 binding to TIM-3 ligand(TIM-3L). In one embodiment, a method includes administering TIM-3,subsequence or fragment (such as extracellular region of TIM-3)conjugated with human Fc recombinant protein to an animal capable ofexpressing human immunoglobulin (e.g., a transgenic mice or transgeniccattle); screening the animal for expression of human TIM-3 antibody;selecting an animal that produces a human TIM-3 antibody; isolating anantibody from the selected animal. In one embodiment, using this method,one can determine whether the human TIM-3 antibody inhibits or preventsTIM-3 binding to TIM-3 ligand (TIM-3L).

As a method for controlling effector activity of the anti-TIM-3monoclonal antibody of the invention, examples include a method whichcontrols the amount of the fucose (also called core fucose) which isbound to N-acetylglucosamine (GlcNAc) through α-1,6 bond in a reducingend of a complex-type N-linked sugar chain which is bound to asparagine(Asn) at position 297 of an Fc region of an antibody (WO2005/035586,WO2002/31140, WO00/61739), a method in which is controlled by modifyingamino acid residues of Fc region of the antibody, and the like. Theeffector activity can be controlled by applying any one of these methodsto the anti-TIM-3 monoclonal antibody of the invention.

The effector activity means an antibody-dependent activity induced viaFc region of antibody, and such as antibody-dependent cellularcytotoxicity (ADCC activity), complement-dependent cytotoxicity (CDCactivity), antibody-dependent phagocytosis (ADP activity) by phagocytessuch as macrophage and dendritic cell, and the like, are known.

By controlling the content of the core fucose of complex-type N-linkedsugar chain of Fc of the antibody, effector activity of the antibody canbe increased or decreased. As a method for reducing the content of thefucose which binds to the complex-type N-linked sugar chain which isbound to Fc of the antibody, defucosylation can be mentioned. Thedefucosylation is to express an antibody using CHO cell from whichα1,6-fucose transferase gene is deleted, and an antibody to which fucoseis not bound can be obtained. The antibody to which fucose is not boundhas high ADCC activity. On the other hand, as a method for increasingthe content of the fucose which binds to the complex-type N-linked sugarchain to which Fc of the antibody is bound, the antibody to which fucoseis bound can be obtained by expressing the antibody using a host cell inwhich α1,6-fucose transferase gene is introduced. The antibody to whichfucose is bound has the ADCC activity lower than that of the antibody towhich fucose is not bound.

In addition, ADCC activity and CDC activity can be increased ordecreased by modifying amino acid residues of Fc of an antibody. Forexample, CDC activity of the antibody can be increased by using theamino acid sequence of the Fc region described in US 2007/0148165. Also,ADCC activity or CDC activity can be increased or decreased by carryingout the amino acid modification described in U.S. Pat. Nos. 6,737,056,7,297,775 and 7,317,091.

Further, an antibody in which effector activity of the antibody iscontrolled can be obtained by using the above-mentioned methods incombination in one antibody.

The nucleic acid may have various lengths. The length of the nucleicacid encoding the TIM-3 antibody of the present invention or thesubsequence thereof is generally about 100 to 600 nucleotides, or anynumerical value or range within encompassing such lengths the abovedescribed range; 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to350, 350 to 400, 400 to 450, 450 to 500, 500 to 550 or 550 to 600nucleotide length, or any numerical value or range or value within orencompassing such length the above described range. Examples of thelength of nucleic acid which specifically hybridize with nucleic acidencoding the TIM-3 antibody of the present invention or the subsequencethereof include generally 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500to 600 nucleotides and any numerical value or range within orencompassing such length.

The terms “nucleic acid” and “polynucleotide” means at least two or moreribo- or deoxy-ribo nucleic acid base pairs (nucleotide) linked whichare through a phosphoester bond or equivalent. The nucleic acid includespolynucleotide and polynucleoside. The nucleic acid includes a singlemolecule, a double molecule, a triple molecule, a circular molecule or alinear molecule. Examples of the nucleic acid include RNA, DNA, cDNA, agenomic nucleic acid, a naturally existing nucleic acid and anon-natural nucleic acid such as a synthetic nucleic acid, but are notlimited. Short nucleic acids and polynucleotides (e.g., 10 to 20, 20 to30, 30 to 50, 50 to 100 nucleotides) are commonly called“oligonucleotides” or “probes” of single-stranded or double-strandedDNA.

The nucleic acid can be prepared using various standard cloningtechniques and chemical synthesis techniques. Examples of the techniquesinclude, but are not limited to, nucleic acid amplification such aspolymerase chain reaction (PCR), with genomic DNA or cDNA targets usingprimers (e.g., a degenerate primer mixture) which can be annealed withan antibody encoding sequence. In addition, nucleic acid can also beprepared by chemical synthesis (e.g., solid phase phosphoamiditesynthesis) or transcription from a gene. The prepared sequence can beexpressed by a cell (e.g., a host cell such as yeast, bacteria oreukaryote (an animal or mammal cell or a plant)) after it is translatedin vitro or cloned into a plasmid and then amplified.

The vector is a vehicle which can be manipulated by insertion orincorporation of nucleic acid. Examples of the vector include a plasmidvector, a virus vector, a prokaryote (bacterium) vector and a eukaryote(plant, fungi, mammals) vector. The vector can be used for in vitro orin vivo expression of nucleic acid. Such a vector is called “expressionvector” and is useful for the transfer of nucleic acid including anucleic acid which encodes an TIM-3 antibody or its subsequence orfragment and the expression of an encoded protein by in vitro (e.g., ina solution or on solid phase), by a cell or by in vivo in a subject.

In addition, the vector can also be used for manipulation of nucleicacids. For genetic manipulation, an inserted nucleic acid can betranscribed or translated using a “cloning vector” in vitro (e.g., in asolution or on solid phase), in a cell or in vivo in a subject.

In general, the vector contains an origin of replication foramplification in a cell in vitro or in vivo. Control elements such as anexpression control element present in the vector can be included inorder to facilitate transcription and translation, if necessary.

A vector may include a selection marker. The “selection marker” is agene which allows for the selection of a cell containing the gene.“Positive selection” means a process for selecting a cell containing theselection marker due to a positive selection. Drug resistance is anexample of the positive selection marker, and a cell containing themarker will survive in culture medium containing the drug and a cellwhich does not contain the marker will die. Examples of the selectionmarker include drug resistance genes such as neo which providesresistance to G418; hygr which provides resistance to hygromycin; purowhich provides resistance to puromycin, and the like. Other positiveselection maker includes genes which enable identification or screeningof a cell containing the marker. Examples of these genes include afluorescent protein (GFP and GFP-like chromophore, luciferase) gene,lacZ gene, alkaline phosphatase gene, and a surface marker such as CD8.“Negative selection” means a process for killing cells which containnegative selection markers by exposing to an appropriate negativeselection agent. For example, a cell containing a herpes simplex virusthymidine kinase (HSV-tk) gene (Wigler et al., Cell, 11: 223 (1977)) issensitive to a drug ganciclovir (GANC). Similarly, gpt gene makes a cellsensitive to 6-thioxantine.

The virus vector includes those which are based on retroviral (alentivirus for infecting not only dividing cells but also non-dividingcells), foamy virus (U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577,6,013,516 and 5,674,703; WO 92/05266 and WO 92/14829), adenovirus (U.S.Pat. Nos. 5,700,470, 5,731,172 and 5,928,944), adeno-associated virus(AAV) (U.S. Pat. No. 5,604,090), a herpes simplex virus vector (U.S.Pat. No. 5,501,979), a cytomegalovirus (CMV) system vector (U.S. Pat.No. 5,561,063), reovirus, rotavirus genome, simian virus 40 (SV40) orpapilloma virus (Cone et al., Proc. Natl. Acad. Sci. USA, 81:6349(1984); Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, editedby Gluzman, 1982; Sarver et al., Mol. Cell. Biol., 1: 486 (1981); U.S.Pat. No. 5,719,054). Adenovirus efficiently infects a slowly replicatingand/or terminally differentiated cell, and can be used to target theslowly replicating cell and/or terminally differentiated cell.Additional examples of virus vectors useful for expression includeparbovirus, Norwalk virus, corona virus, paramyxo virus and rhabdovirus, toga virus (e.g., Sindobis virus and Semliki forest virus) andvesicular stomatitis virus (VSV).

A vector comprising a nucleotide acid can be expressed when the nucleicacid is connected to expression elements so as to function. The term“connected so as to function” (operably linked) means that a physical orfunctional relation between the elements referred to that permit them tooperate in their intended fashion. Accordingly, the nucleic acid“operably linked” to an expression control element means that thecontrol element modulates nucleic acid transcription and, asappropriate, translation of the transcription product.

The “expression control element” or “expression control sequence” is apolynucleotide which influences upon expression of an operably linkednucleic acid. Promoters and enhancers are non-limiting specific examplesof expression controlling elements and sequences. The “promoter” is acis-acting DNA regulatory region which can initiate transcription ofdownstream (3′ direction) nucleic acid sequence. A nucleotide whichaccelerates transcription initiation is included in the promotersequence. The enhancer also regulates nucleic acid expression but actsat a distance from the transcription initiation site of the nucleic acidto which it is operably linked. When the enhancer is present in either5′ or 3′ end of the nucleic acid as well as within the nucleic acid(e.g., intron or coding sequence), the enhancer further functions.Additional examples of the expression control element include a leadersequence and a fusion partner sequence, an internal ribosome entry site(IRES) element for preparing multigene, or polycistronic message,splicing signal of intron, maintenance of correct reading frame of geneto enable inframe translation of mRNA, polyadenylation signal whichproduces proper polyadenylation of the transcription product ofinterest, and stop codons.

Examples the expression control element include a “constitutional”element in which transcription of an operably linked nucleic acid occurswithout the presence of signals or stimulus. The expression controlelement which confers expression in response to the signal or stimulusand increase or decrease expression of the operably linked nucleic acidis “adjustable”. The adjustable element which increases expression ofthe operably linked nucleic acid in response to a signal or stimulus iscalled an “inducible element”. The adjustable element which decreasesexpression of the operably linked nucleic acid in response to a signalor stimulus is called “repressor element” (namely, the signal decreasesthe expression; and the expression increases when the signal is removedor not present).

Examples of the constitutional promoter for bacterial expression includean inducing promoter, such as T7 and pL, plac, ptrp and ptac (ptrp-lachybrid promoter) of bacteriophage λ and the like. For insect cellsystem, a constitutional or inducible promoter (e.g., ecdysone) can beused. The constitutional promoter for yeast, include an inducingpromoter such as ADH, LEU2, GAL and the like (e.g., see Ausubel et al.,In: Current Protocols in Molecular Biology, Vol. 2, Chapter 13, GreenePublish. Assoc. & Wiley Interscience edition, 1988; Grant et al., In:Methods in Enzymology, 153: 516-544 (1987) Wu & Grossman, 1987, Acad.Press, N.Y.; Glover, DNA Cloning, Vol. 11, Chapter 3, IRL Press, Wash.,D.C., 1986; Bitter, In: Methods in Enzymology, 152: 673-684 (1987),edited by Berger & Kimmel, Acad. Press, N.Y.; and Strathern et al., TheMolecular Biology of the Yeast Saccharomyces, edited by Cold SpringHarbor Press, Vol. 1 and Vol. 11 (1982)).

For the expression in mammals, a constitutional promoter derived from avirus or other origin can be used. For example, inducible promotersderived from CMV, SV40, or a viral long terminal repeated sequence(LTR), or mammal cell genome (e.g., metallothionein IIA promoter; heatshock promoter, steroid/thyroid hormone/retinoic acid respondingelement) or mammal virus (e.g., adenovirus late promoter; mouse breastcancer virus LTR) can be used.

Examples of the expression control element include an element which isactive in a specific tissue or cell types, and such an element is called“tissue specific expression control element”. In general, the tissuespecific expression control element is more active in specific cells ortissue types, and this is because this tissue specific expressioncontrol element is recognized by a transcription activating proteinwhich is active in the specific cell or tissue types or by othertranscription factor, as compared to other cells or tissue types.Non-limiting specific examples of such an expression control element arehexokinase II, COX-2, α-fetoprotein, carcinoembryonic antigen, DE3/MUC1,prostate specific antigen, C-erB2/neu, glucose-dependent insulinsecretion stimulatory polypeptide (GIP), telomerase reversetranscriptase and a promoter such as hypoxia-responsive promoter.

According to the invention, a host cell transformed or transfected withTIM-3 nucleic acid or vector is provided. Examples of the host cells,but are not limited to, include prokaryotic cell and eukaryotic cell,such as, bacteria, fungi (yeast), and cells of plants, insects andanimals (e.g., mammals such as primates, human and the like).Non-limiting examples of transformed cell include a bacteria transformedwith a recombinant bacteriophage nucleic acid, a plasmid nucleic acid orcosmid nucleic acid expression vector; a yeast transformed with arecombinant yeast expression vector; a plant cell infected with arecombinant virus expression vector (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with a recombinantplasmid expression vector (e.g., Ti plasmid); an incest cell infectedwith a recombinant virus expression vector (e.g., baculovirus); and ananimal cell infected with a recombinant virus expression vector (e.g.,retrovirus, adenovirus, vaccinia virus) or a transformed animal cellmanipulated for stable expression. A CHO cell is a non-limiting exampleof a mammal host cell which expresses a TIM-3 antibody and itssubsequence thereof and fragment. The host cell may be a plurality orpopulation of cells from a primary cell isolated strain, an isolatedsecondary cell or subcultured cell, or an established cell line orimmortalized cell culture.

The term “be transformed” or be transfected” when it is used inreference to a cell (e.g., host cell) or an organism means a change ofgene in a cell after incorporation of an exogenous molecule, such as aprotein or a nucleic acid (e.g., transgene), into the cell. Accordingly,the “transfected” or “transformed” cell is a cell into which theexogenous molecule is introduced by the hand of man, for example,recombinant DNA techniques or a progeny thereof.

The nucleic acid or protein can be transfected or transformed(expressed) in the cell or a progeny thereof stably or temporarily. Theintroduced protein can be expressed by growing the cell, or transcribingthe nucleic acid. Since there is a possibility that a mutation occursduring replication, there is a case that a progeny of the transfected ortransformed cell is not identical to the parent cell.

In general, a vector is used in the cell transfection or transformation.The vector can be included in a viral particle or vesicle and can beoptionally directed demands to a specific cell types by including aprotein on the particle or vesicle surface which binds to a target cellligand or receptor. Accordingly, a cell can be used as a target bypreparing the viral particle or vesicle itself or the viral surfaceprotein, for the purpose of an in vitro, ex vivo or in vivo transfectionor transformation. Therefore, the vector includes in vitro, in vivo andex vivo delivering techniques of viral and non-viral vectors into acell, tissue or organ.

In addition, introduction of a nucleic acid into a target cell (e.g., ahost cell) can also be carried out by a method conventionally known inthe technical field, such as osmotic shock (e.g., calcium phosphate),electroporation, microinjection, cell fusion and the like. Theintroduction of nucleic acid and polypeptide in vitro, ex vivo and invivo can also be carried out using other techniques. For example, apolymer substance such as polyester, poyamic acid, hydrogel, polyvinylpyrrolidone, ethylene-vinyl acetate, methyl cellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymer, polylactide/glycolide copolymer, or ethylene vinyl acetatecopolymer and the like can be used. The nucleic acid can be enclosed ina microcapsule using a hydroxymethyl cellulose or gelatin-microcapsuleor poly(methyl methacrylate)-microcapsule, or a colloid system,respectively, by a coacervation technique or by interfacialpolymerization. The colloid dispersion system includes a system based ona polymer complex, nanocapsule, microsphere, beads and lipid(oil-in-water type emulsion, micelle, mixed micelle, liposome and thelike).

The liposome for introducing various compositions into cells isconventionally known in the technical field, and for example,phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP areincluded therein (e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740and 4,975,282; and GIBCO-BRL, Gaithersburg, Md.). Piperazine basedamphilic cationic lipids which is useful in gene therapy (see e.g., U.S.Pat. No. 5,861,397) are also known. A cationic lipid system is alsoknown (see e.g., U.S. Pat. No. 5,459,127). In this specification, thepolymer substance, microcapsule and colloid dispersion system (liposomeand the like) are collectively called as “vesicle”.

In addition, examples of the suitable techniques which can be used inthe method for an antibody are affinity purification, non-modified gelpurification, HPLC or RP-HPLC, size exclusion, purification by protein Acolumn and an optional combination of these techniques. An anti-TIM-3antibody isotype can be determined using ELISA assay, and for example,human Ig can be identified using mouse Ig absorbed anti-human Ig.

The binding affinity can be determined by association (Ka) anddissociation (Kd) rates. The equilibrium affinity constant KD is theratio of Ka/Kd. The association (Ka) and dissociation (Kd) rates can bemeasured using surface plasmon resonance (SPR) (Rich and Myszka, Curr.Opin. Biotechnol., 11: 54 (2000): Englebienne, Analyst., 123: 1599(1998)). Instrumentation and methods for real time detection andmonitoring of association rate are conventionally known and commerciallyavailable (BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist,Biochem. Soc. Trans., 27:335 (1999)). The KD value can be defined as theTIM-3 antibody concentration required to saturate one half of thebinding site (50%) on TIM-3.

(Pharmaceutical Composition)

Antibodies can be included in a pharmaceutical composition. In anembodiment, an antibody comprises a pharmaceutically acceptable carrier,a stabilizer or a filler and is prepared in the form of aqueous solutionor as a freeze-dried preparation. Typically, an appropriate amount of apharmaceutically acceptable salt is used for isotonicity of thepharmaceutical preparation. Examples of the acceptable carrier,stabilizer or filler include a buffer solution such as phosphate,citrate and other organic acid and the like; a low molecular weight(less than 10 in the number of residues) polypeptide; a protein such asserum albumin, gelatin, immunoglobulin and the like; a hydrophilicpolymer such as polyvinyl pyrrolidone; an amino acid such as glycine,glutamine, asparagine, histidine, arginine, lysine and the like; amonosaccharide such as glucose, mannose, dextrin and the like,disaccharides and other carbohydrates; a chelating agent such as EDTAand the like; saccharides such as sucrose, mannitol, trehalose, sorbitoland the like; a salt forming counter ion such as sodium and the like; anantioxidant including methionine and ascorbic acid; a metal complex(e.g., Zn-protein complex); an antiseptic (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; banzalconiumchloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol; and m-cresol); and/or a nonionic surfactantsuch as TWEEN™, PLURONICS™, polyethylene glycol (PEG) and the like.

(Therapeutic Use of Antitumor Substance which Target TIM-3 ExpressionCells)

Examples of the antitumor substances targeting TIM-3 expressing cellsare anti-TIM-3 antibodies, but are not limited to.

Examples of the diseases for which the therapeutic use is examined, butare not limited thereto, include the diseases which can be considered totreat by binding or targeting TIM-3-expressing blood tumor cells (AMLcell, CML cell, MDS cell, ALL cell, CLL cell, Multiple Myeloma cell andthe like), helper T cell (e.g., Th1 cell, Th17 cell) antigen presentingcell (e.g., dendritic cell, monocyte macrophage and related cells(hepatic stellate cell, osteoclast, microglia, intraepidermalmacrophage, dust cell (alveolar phagocyte) and the like)). Examples ofthe disease for which therapeutic use is examined include a blooddisease, especially hematologic tumor, in which expression of TIM-3 isfound in bone marrow or peripheral blood. Specific example may includeacute myelocytic leukemia AML). Based on the FAB classification(French-American-British criteria) which can determine which stage ofthe cell among the cells in the course of differentiating into variousblood cells from the hematopoietic stem cell caused tumorigenictransformation, the acute myelocytic leukemia is classified into diseasetypes of M0 (micro-differentiation type myeloblastic leukemia), M1(undifferentiated myeloblastic leukemia), M2 (differentiatedmyeloblastic leukemia), M3 (acute promyelocytic leukemia), M4(myelomonocytic leukemia), M5 (monocytic leukemia), M6(erythroleukemia), M7 (megakaryocytic leukemia) and subtypes thereof. Inaddition, further examples of diseases include acute lymphocyticleukemia, atypical leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, adult T cell leukemia, NK/T cell lymphoma,granular lymphocytosis (LGL leukemia), polycythemia vera, essentialthrombocythemia, hypereosinophilic syndrome, myelodysplastic syndrome,lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma, B-celllymphoma (such as follicular lymphoma, MALT lymphoma, mantle celllymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblasticlymphoma and Catsleman disease), T cell lymphoma (T/NK cell lymphoma,adult T cell leukemia and NK/T cell lymphoma).

The method of the invention which comprises administration or deliveryof an anti-TIM-3 antibody and an anti-tumor substance which targets aTIM-3 expression cell can be carried out by any acceptable method. In aspecified embodiment, these are administered to a subject, locally,regionally or systemically.

In addition, regarding the TIM-3 antibody or the antitumor substancewhich targets TIM-3 expression cell for treating the above-mentioneddiseases can also be considered to combine with other therapeutic agentsuitable for the same disease (typically a chemotherapeutic agent) or beadministered in combination with radiotherapy.

Examples of the suitable other therapeutic agent include achemotherapeutic agent such as cytarabine (Ara-C), an anthracyclinesystem antitumor agent (typically, daunorubicin (DNR), idarubicin (IDA))and the like, a differentiation inducing therapeutic agent such asall-trans retinoic acid (ATRA), arsenious acid, and Am80 (tamibarotene),gemtuzumab-ozogamicin (ozogamicin conjugate anti-CD33 antibody),topotecan, fludarabine, cyclosporine, mitoxantrone (MIT), interferon andimatinib, but are not limited thereto, and also include a combinationwith a therapeutic method considered to be clinically effective.

Mammals (e.g., human) are included in the subject which can be treatedby the invention. In a specified embodiment, it is a subject who is acandidate of blood tumor or who received treatment of the blood tumor, asubject who is a candidate of a blood tumor and in whom TIM-3 expressingcells are detected, a subject having a possibility causingTIM-3-mediated cellular response or who received treatment of theTIM-3-mediated cellular response, a subject who is a candidate of amyelocytic malignant tumor or who received treatment of the myelocyticmalignant tumor or a subject who is a candidate of acute myelocyticleukemia or who received treatment of the acute myelocytic leukemia.

According to this specification, the terms “treat”, “treating”,“treatment” and the grammatical variations thereof mean a protocol, aplanning, a process or an improving method which is carried out on eachsubject who is desirable to obtain physiological effect or good outcomeon the patient. Accordingly, the method of the invention includes atreatment and a treating method which produce measurable improvement orbeneficial effect, particularly on a disorder, a disease, pathology, acondition of a disease or a symptom of a given subject. The measurableimprovement or profitable effect is objective or subjective, transientor long-term improvement of any one of disorders, diseases,phisiological conditions, conditions of a disease or symptoms, or areduction in onset, severity, duration or frequency of adverse symptomrelated to or caused by disorders, diseases, physiological conditions,conditions of a disease or state. According to the method of theinvention, there is a possibility that its effect is not alwaysexhibited immediately, but eventual improvement or beneficial effect isfound a little later with the lapse of time, so that stabilization oramelioration in a give subject will occur.

In the specification, the term “recurrence” refers to the condition inwhich symptoms worsen or a subject gets into non-remission state afterthe therapy is once successful or induces remission in terms ofhematology. The “remission” in leukemia refers to the conditions inwhich any blast of leukemia cells are not found in peripheral blood. Theproposed remission state in acute myeloid leukemia is defined by NIH(see Cheson B D, et al., Journal of Clinical Oncology, Vol. 8, 813-819).In addition, the term “refractory” refers to the condition in whicheffects due to treatment are not found in a subject.

(Antagonist)

Antibodies further include those that affect a function or activity ofTIM-3. In particular embodiments, an antibody inhibits or prevents thebinding of TIM-3 ligand to TIM-3; inhibits or prevents the binding ofTIM-3 ligand to cell; modulate TIM-3-mediated cell signaling (e.g.,inhibits or prevents); modulate TIM-3-mediated cell response. Inparticular aspects, a TIM-3-mediated cell response comprisesproliferation of TIM-3 expressing cell, promotion of production ofcytokine such as IFNγ and enhancement of tumor immunity.

(Use of Agonist Antibody)

Antibodies further include those that affect a function or activity ofTIM-3. In particular embodiments, an antibody mimic the binding of TIM-3ligand to TIM-3; modulate (e.g., promote or enhance) TIM-3-mediatedsignal transduction; modulate TIM-3-mediated cell response. The TIM-3expressing cell response induces growth arrest of TIM-3 expressing cell,controlling of production of cytokines and the like.

Unless otherwise noted, all of the technical terms and scientific termsused in this specification have the same meanings of those which aregenerally evident for persons in the technical field to which theinvention is related. Methods and materials similar or equivalent tothose described in this specification can be used in the operations orexaminations of the invention, but those which are described in thisspecification are suitable methods and materials.

EXAMPLES Example 1 Preparation of Bone Marrow and Peripheral Blood Cells

Provision of the analytes from patients and healthy volunteers wascarried out under the recognition by an ethical committee at the KyushuUniversity Hospital. The bone marrow cell was collected by a bone-marrowaspiration from a patient of leukemia or myelodysplastic syndrome and ahealthy volunteer. The peripheral blood was collected by an intravenousblood collection from a healthy volunteer. The stem cell-mobilizedperipheral blood was collected from a patient indicated for autologousperipheral blood stem cell transplantation (a case in which a patienthas a relapse of a diffuse large cell type B cell lymphoma afterentering into complete remission with chemotherapy and has sensitivityfor the chemotherapy). Specifically, after completion of chemotherapy(Cyclophosphamide (CY) massive dose therapy or VP-16 (etoposide) massivedose therapy), peripheral blood stem cells were mobilized by theadministration of a G-CSF preparation (Filgrastim), GRAN® (Kyowa HakkoKirin Co., Ltd.) and collected using a standard apheresis. A portion ofmononuclear cells collected to confirm the existance of CD34 positivecells was used for experiment. In this connection, the dose and scheduleof drugs were standard protocol covered by insurance.

Heparin was added to the collected cells to prevent coagulation. Afterdilution of each analyte with PBS, Ficoll-Plaque Plus (GE Healthcare)was spread under it. The mononuclear cells were separated from serum andplatelets by carrying out centrifugation at 1700 rpm for 30 minutes (abrake was not applied). The boundary containing PBMC was collected andwashed using a staining medium. After suspension with the stainingmedium again, the cells were mixed with Türk solution and then counted.The cells were re-suspended using 100 μl of the staining medium per1×10⁶ cells. To stain normal hematopoietic stem cells and precursorcells, positive selection of CD34 positive cells was carried out usingIndirect CD34 MicroBead Kit (Miltenyi Biotec) after the above-mentionedmononuclear cell separation and then the staining medium was added togive a final volume of 100 μl.

Example 2 Staining and Analyzing Methods of Bone Marrow NormalHematopoietic and Tumor Stem Cells

The cells were stained using 2 μl of an anti-human CD34 antibody(manufactured by Becton Dickinson & Co., (hereinafter referred to asBD), Cat No. 340441), 20 μl of anti-human CD38 antibody (manufactured byCALTAG, Cat No. MHCD3815), 2 μl of anti-human CD90 antibody(manufactured by BD, Cat No. 555595) or 20 μl of anti-TIM-3 antibody(manufactured by R & D Systems, 344823) as the primary antibodies at 4°C. for 40 minutes. Then, PBS was added for wash, and the obtainedsuspension was centrifuged at 1500 rpm for 5 minutes. After thesupernatant was discarded, 100 μl of the staining medium was addedthereto. The obtained cells were stained by adding 5 μl of each ofanti-human Lineage antibodies (anti-human CD3 (manufactured by BD, CatNo. 555341), CD4 (manufactured by BD, Cat No. 555348), CD8 (manufacturedby BD, Cat No. 555636), CD10 (manufactured by BD, Cat No. 555376), CD19(manufactured by BD, Cat No. 555414), CD20 (manufactured by BD, Cat No.555624), CD11b (manufactured by BD, Cat No. 555389), CD14 (manufacturedby Beckman Coulter, Cat No. A07765), CD56 (manufactured by BD, Cat No.555517) and GPA (manufactured by BD, Cat No. 559944) antibodies as thesecondary antibody, and 5 μl of streptoavidin APC-Cy7 (manufactured byBD, Cat No. 554063) at 4° C. for 40 minutes. After washing with PBS, theobtained cells were re-suspended with PI-containing staining medium.Analysis and sorting of the analytes were carried out by FACSAria (BD).

Example 3 Expression of Human TIM-3 Molecule in Human Blood Tumor

Expression of human TIM-3 molecule in human blood tumor was examinedusing the methods of Example 1 and Example 2.

Expression of human TIM-3 molecule in AML (M0) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 1 and Table 1. In the patientswith AML (M0), expression of TIM-3 was observed in one of the two cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M0) cells.

Expression of human TIM-3 molecule in AML (M1) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 2 and Table 1. In the patientswith AML (M1), expression of TIM-3 was observed in all nine cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M1) cells.

TABLE 1 % of TIM-3(+) cells Cytogenetic Lin(−)CD34(+) Lin(−)CD34(+)Lin(−) Pt FAB Abnomality CD38(−) CD38(+) CD34(−) AML1 M2 t(8;21) 89.485.7 45 AML2 M2 46XY 49.7 94.7 44.6 AML3 M4 46XY 86 84.4 82.4 AML4 M246XX 83.3 90.6 81.8 AML5 M0 4.2 1.1 4.4 AML6 M2 77.5 82.9 30 AML7 M566.7 58.4 82.2 AML8 M2 60.6 89 62.1 AML9 M2 96.5 91 64 AML10 M4 99.698.5 71.2 AML11 M3 PML/RARa 1.6 22.7 52.2 AML12 M1 46XY 64.4 73.5 36.7AML13 M2 t(8;21) 65.7 69 43.9 AML14 M1 26.6 23.3 19 AML15 M1 50.3 69.150.8 AML16 M4 inv 16 93.7 94.2 30.1 AML17 M2 91.2 93.5 36.1 AML18 M5 2.337.7 80.3 AML19 M6 18 34.6 37.6 AML20 M1 Flt3-ITD+ 74 76.3 32 AML21 M1Flt3-ITD+ 82.2 90.5 85 AML22 M1 97.2 98 93.1 AML23 M2 94.2 94.7 40 AML24M0 97.8 99.4 69.6 AML25 M1 38.8 65.8 33 AML26 M1 72.5 83.1 34.9 AML27 M4Flt3-ITD+ 84.9 90.1 78 AML28 M1 74.6 77.7 33.4 AML29 M2 98.3 99.1 79.2AML30 M6 35.5 53.6 24.5 AML31 M4 56.1 68.5 77.7

Expression of human TIM-3 molecule in AML (M2) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 3 and Table 1. In the patientswith AML (M2), expression of TIM-3 was observed in all ten cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M2) cells.

Expression of human TIM-3 molecule in AML (M3) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 4 and Table 1. In the patientwith AML (M3), expression of TIM-3 was observed in one case.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M3) cells.

Expression of human TIM-3 molecule in AML (M4) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 5 and Table 1. In the patientswith AML (M4), expression of TIM-3 was observed in all five cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M4) cells.

Expression of human TIM-3 molecule in AML (M5) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 6 and Table 1. In the patientswith AML (M5), expression of TIM-3 was observed in all two cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M5) cells.

Expression of human TIM-3 molecule in AML (M6) patient-derived bonemarrow Lin(−)CD34(+)CD38(−) cell, Lin(−)CD34(+)CD38(+) cell andLin(−)CD34(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 7 and Table 1. In the patientswith AML (M6), expression of TIM-3 was observed in all two cases.Accordingly, this example showed usefulness of TIM-3 as a therapeutictarget for AML (M6) cells.

Expression of human TIM-3 molecule in MDS patient-derived bone marrowLin(−)CD34(+)CD38(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 9. In the patients with MDS,expression of TIM-3 was observed in all seven cases.

Expression of human TIM-3 molecule in CML patient-derived bone marrowLin(−)CD34(+)CD38(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 10. In the patients with CML,expression of TIM-3 was observed in all two cases.

Expression of human TIM-3 molecule in ALL patient-derived bone marrowLin(−)CD34(+)CD38(−) cell was examined by analysis using multicolor flowcytometry. The results are shown in FIG. 11. In the patient with ALL,expression of TIM-3 was not observed in one case.

Expression of human TIM-3 molecule in bone marrow Lin(−)CD34(+)CD38(−)cell and Lin(−)CD34(+)CD38(+) cell derived from patients with recurrentAML was examined by analysis using multicolor flow cytometry. Thetypical results are shown in FIG. 12. In the patients with recurrentAML, expression of TIM-3 was observed in Lin(−)CD34(+)CD38(−) leukemiastem cell and Lin(−)CD34(+)CD38(+) AML cell.

Example 4 Expression of Human TIM-3 Molecule in Human Normal Blood Cells

The methods were the same as the preparation of bone marrow andperipheral blood cells in Example 1 and the staining of bone marrownormal hematopoietic and tumor stem cells and analyzing method inExample 2.

Expression of human TIM-3 molecule in Lin(−)CD34(+)CD38(−) cell andLin(−)CD34(+)CD38(+) cell derived from bone marrow and mobilizedperipheral blood stem cells of healthy volunteer was examined by themulticolor flow cytometry analysis. The results are shown in FIG. 13.Expression of the TIM-3 was not observed in theLin(−)CD34(+)CD38(−)CD90(+) cell which is a normal hematopoietic stemcell fraction.

In addition, it was observed that TIM-3 was partially expressed incommon lymphoid progenitor cells (CLP), common myeloid progenitor cells(CMP) and Granulocyte-Monocyte Progenitor cells (GMP), but was notexpressed in Megakaryocyte-Erythroid Progenitor cells (MEP).

Expression of human TIM-3 molecule in healthy volunteer-derived normalperipheral blood cells was examined by the flow cytometry analysis. Inaccordance with the preparation of bone marrow and peripheral bloodcells of Example 1, the cells were stained using 2 μl of anti-CD3antibody (manufactured by BD, Cat No. 555339), 2 μl of anti-CD14antibody (manufactured by BD, Cat No. 555413), 10 μl of anti-CD19antibody (manufactured by BD, Cat No. 555399) and 20 μl of anti-TIM-3antibody (manufactured by R & D Systems, 344823) at 4° C. for 40minutes. After washing with the staining medium, the cells werere-suspended with PI-added staining medium. The analytes were analyzedby FACSAria (BD).

The results are shown in FIG. 14. Clear expression of TIM-3 was observedin the monocyte. Expression of TIM-3 was also observed in a part of theCD3 positive T cells. Its expression was not observed in thegranulocytes and B cells.

Example 5 Expression of TIM-3 in Cell Lines

Expression of hTIM-3 in human cell lines was analyzed by flow cytometry

The cells in culture were harvested by pipetting and concentrated bycentrifugation. After washing with the staining medium, blocking wascarried out using human IgG (manufactured by SIGMA, final concentration1 mg/ml). The cells were stained with a PE-labeled anti-human TIM-3monoclonal antibody (R & D Systems) and allowed to stand still at 4° C.for 30 minutes. After adding 7-AAD (BD Biosciences) and washing with thestaining medium, the resulting cells were resuspended in the stainingmedium and analyzed by FACS Calibur (Becton, Dickinson and Company). Theresults are shown in FIG. 15. Expression of TIM-3 was observed in theMultiple Myeloma-derived RPMI-8226 cells and ARH77 cells, the B celllymphoma-derived Daudi cells, the T cell lymphoma-derived SR-786 cellsand the NK cells lymphoma-derived NK-92 cells. In addition, expressionof TIM-3 was also observed in the AML-derived KG-1 cells.

For this reason, it was shown a possibility that TIM-3 was expressed inMultiple Myeloma and AML and showed that the anti-human TIM-3 antibodywas useful as a therapeutic agent for these diseases.

Example 6 Preparation of Human Fc Fusion Protein of Soluble Form ofExtracellular Human TIM-3 and Soluble Form of Extracellular Human TIM-3Protein

The protein is prepared according to the following method.

(Molecular Cloning of hTIM-3 cDNA)

hTIM-3 cDNA was amplified from a leukocyte-derived cDNA (CLONTECH HumanMTC Panel) by PCR using ExTaq (Takara Bio Co. Ltd.). As a PCR device,GeneAmp PCR System 9700 (Applied Biosystems, hereinafter, the PCR deviceis the same in this specification) was used. Regarding the PCR, after adenaturation step at 95° C. for 1 minute, a three step reaction at 95°C. 15 seconds-58° C. 15 seconds-72° C. 30 seconds was carried out 40cycles and then a reaction at 72° C. for 2 minutes was carried out. ThePCR primers used were as follows.

TIM-3 Fw2: (SEQ ID NO: 3) 5′-GCCACCATGTTTTCACATCTTCCCTT-3′ TIM-3 Re2:(SEQ ID NO: 4) 5′-CTATGGCATTGCAAAGCGAC-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band at around 0.9 kb was cut out andextracted using Wizard SV Gel and PCR Clean-Up System. Then 4.5 μL ofthe extracted DNA was mixed with 0.5 μL of pGEM-T Easy vector (Promega)and ligated using TaKaRa Ligation Kit. Regarding the transformation, theligation sample and a DH10B competent cell were mixed and spread on LBplate (containing X-gal and ampicillin). Insert check of pGEM-T Easyvector was carried out by colony direct PCR using LA Taq (Takara Bio Co.Ltd.). Regarding the PCR, after a denaturation step at 95° C. for 1minute, a three step reaction at 95° C. 15 seconds-56° C. 15 seconds-72°C. 30 seconds was carried out 35 cycles and then a reaction at 72° C.for 2 minutes was carried out. The primers used were as follows.

T7: (SEQ ID NO: 5) 5′-TAATACGACTCACTATAGGG-3′ SP6: (SEQ ID NO: 6)5′-CATACGATTTAGGTGACACTATAG-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ataround 1.2 kb was obtained, nucleotide sequence was determined by adirect sequencing method. Sequence reaction was carried out in GeneAmpPCR System 9700 (Applied Biosystems) using BigDye® Terminator v3.1 CycleSequencing Kit (Applied Biosystems) (hereinafter, reagent or device ofthe DNA sequence analysis is the same in this specification). As the PCRprimers, T7 and SP6 were used. As the sequence analyzer, ABI 3700XL DNAanalyzer (Applied Biosystems) was used (hereinafter, device of the DNAsequence analysis is the same in this specification). A clone which havethe same sequence of the coding region of GenBank accession numberNM_032782 was selected and its plasmid DNA was extracted by the Miniprepmethod.

(Preparation of hTIM-3 Expression Vector)

For preparation of hTIM-3 expression vector, the clone in which 5′terminal of human TIM-3 cDNA was at T7 side in cloning site of pGEM-TEasy vector. hTIM-3/GEM-T Easy plasmid DNA and pMCs-IGRetrovirus Vector(Cosmobio) were digested with NotI and subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Bands around 0.9 kb and 5 kb were cut out andextracted using Wizard SV Gel and PCR Clean-Up System, respectively.Then 4.5 μL of the extracted hTIM-3 DNA was mixed with 0.5 μL ofpMCs-IGvector DNA and ligated using TaKaRa Ligation Kit. Regarding thetransformation, the ligation sample and a DH10B competent cell weremixed and spread on LB plate (containing ampicillin). Insert check wascarried out by colony direct PCR using LA Taq (Takara Bio Co. Ltd.).Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 45seconds was carried out 35 cycles and then a reaction at 72° C. for 2minutes was carried out.

The primers used were as follows.

pMCs-Fw: (SEQ ID NO: 7) 5′-TCAAAGTAGACGGCATCGCAG-3′ TIM-3 Re1:(SEQ ID NO: 8) 5′-GCATTGCAAAGCGACAAC-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. From a colony in which amplification ataround 1.1 kb was obtained, the plasmid DNA was extracted using Miniprepmethod. The purified hTIM-3/pMCs-IG plasmid DNA was found to have thesame sequence of the coding region of GenBank accession number NM_032782by DNA sequence analysis. The primers used for DNA sequence analysiswere as follows.

pMCs-Fw: (SEQ ID NO: 9) 5′-TCAAAGTAGACGGCATCGCAG-3′ hTIM-3 Fw1:(SEQ ID NO: 10) 5′-ACTCTGGAGCAACCATCA-3′

In order to confirm the expression of hTIM-3 protein, hTIM-3/pMCs-IGplasmid DNA was expressed transiently in 293T cells. FuGene6 (Roche) wasused for transfection. Two days after, 293T cells were harvested andthen washed with a staining medium (PBS containing 2% FCS and 0.05%NaN₃) and stained with PE-labeled anti-human TIM-3 monoclonal antibody(R&D System). After washing with the staining medium, 7-AAD (BDBiosciences) was added and then the obtained cells were analyzed withFACS Calibur (Becton, Dickinson and Company). As a result, expression ofhTIM-3 protein from hTIM-3/pMCs-IG vector was confirmed.

(Preparation of hTIM-3 Stable Expression Cell)

hTIM-3/pMCs-IG or EmptypMCs-IG and VSV-G expression vectors wereintroduced into 293gp cells. FuGene6 (Roche) was used for transfection.Three days after, the culture supernatant was harvested and contaminantswere removed using 0.45 μm filter (Milipore). After centrifugation(6000×g, 4° C. for 7 hours), the precipitate was dissolved with IMDMmedium (Invitrogen). The concentrated retrovirus solution and Protaminesolution (Wako Pure Chemical Industries, Ltd., final concentration: 100μg/ml) were added to the medium of EoL-1 cells and Jarkat cells. Afterseveral passages, GFP positive infected cells in the culture medium weresorted and collected by FACSAria. After further several passages, GFPpositive infected cells in the culture medium were sorted and collectedagain by FACSAria. After several passages, the expression of TIM-3 wasconfirmed by a flow cytometry method using PE-labeled anti-TIM-3monoclonal antibody.

(Preparation of Expression Vector of Fusion Protein of Human Fc Proteinand Soluble Form of Extracellular Human TIM-3)

A cDNA encoding the extracellular region of human TIM-3 was amplified byusing a PCR method and FLAG tag and human Fc sequence were fused to itsdownstream (sTIM-3-FLAG-Fc/pTracerCMV).

The cDNA encoding the extracellular region of human TIM-3 was amplifiedby using a PCR method using hTIM-3/pMCs-IG plasmid as a template andPrimeSTAR® HS DNA Polymerase (Takara Bio Co. Ltd.). Regarding the PCR, atwo step reaction at 98° C. 10 seconds-68° C. 40 seconds was carried out20 cycles. The primers used were as follows.

pMCs-Fw: (SEQ ID NO: 11) 5′-TCAAAGTAGACGGCATCGCAG-3′ TIM3ED-FcReXba:(SEQ ID NO: 12) 5′-TTTTCTAGATCTGATGGTTGCTCCAGA-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band of around 0.6 kb was cut out and theDNA was extracted using Wizard SV Gel and PCR Clean-Up System. Thuspurified DNA was digested with EcoRI and XbaI and subjected to 0.8%agarose gel electrophoresis (135 V, 15 minutes, TAE buffer). A band ofaround 0.9 kb was cut out and the DNA was extracted using Wizard SV Geland PCR Clean-Up System. The obtained DNA was mixed with apTracer-CMV-FLAG-humanFc vector (a plasmid in which FLAG and Fc regionof human IgG were inserted between XbaI site and ApaI site of themodified pTracer-CMV [manufactured by Invitrogen]), which had beencleaved using the same enzymes of the purified DNA, and ligated usingTaKaRa Ligation Kit. Regarding the transformation, the ligation sampleand a DH10B competent cell were mixed and spread on LB plate (containingampicillin). Insert check was carried out by colony direct PCR using LATaq (Takara Bio Co. Ltd.). Regarding the PCR, after a denaturation stepat 95° C. for 1 minute, a three step reaction at 95° C. 15 seconds-56°C. 15 seconds-72° C. 40 seconds was carried out 35 cycles and then areaction at 72° C. for 2 minutes was carried out. The primers used wereas follows.

T7: (SEQ ID NO: 13) 5′-TAATACGACTCACTATAGGG-3′ TIM3ED-FcReXba:(SEQ ID NO: 14) 5′-TTTTCTAGATCTGATGGTTGCTCCAGA-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A plasmid DNA was extracted by the Miniprepmethod from a colony in which amplification of around 0.7 kb wasobtained. It was found by a DNA sequence analysis that the purifiedTIM-3-FLAG-Fc/pTracerCMV plasmid DNA had the sequence identical to thecorresponding region of GenBank accession number NM_032782. The primersused for the DNA sequence analysis were as follows.

T7: (SEQ ID NO: 15) 5′-TAATACGACTCACTATAGGG-3′ hTIM-3 Fw1:(SEQ ID NO: 16) 5′-ACTCTGGAGCAACCATCA-3′

The sequence of the insert (from the following EcoRI recognition site tojust before ApaI recognition site) was as follows.

(SEQ ID NO: 17) GATTGCCACCATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGATATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGATTGGCCAATGACTTACGGGACTCTGGAGCAACCATCAGATCTAGAGCAGACTACAAGGACGACGATGACAAGACTAGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGCGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGATGA(Preparation of Expression Vector of Soluble Form of ExtracellularRegion of Human TIM-3)

A cDNA encoding the extracellular region of human TIM-3 was amplified byusing a PCR method, and FLAG tag was connected to its downstream(sTIM-3-FLAG/pEF6 Myc_HisC).

The cDNA encoding the extracellular region of human TIM-3 was amplifiedby using a PCR method using hTIM-3/pGEM-T Easy plasmid as a template andPrimeSTAR® HS DNA Polymerase (Takara Bio Co. Ltd.). Regarding the PCR, atwo step reaction at 98° C. 10 seconds-68° C. 30 seconds was carried out25 cycles. The primers used were as follows.

TIM-3 Fw2: (SEQ ID NO: 18) 5′-GCCACCATGTTTTCACATCTTCCCTT-3′TIM3ED-FLAG4aa: (SEQ ID NO: 19) 5′-GTCCTTGTAGTCTCTGATGGTTGCTCCAGA-3′

Using 2 μL of the obtained PCR product as a template, the DNA wasamplified by using LA taq (Takara Bio Co. Ltd.) by a PCR method.Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-58° C. 15 seconds-72° C. 30seconds and then a reaction at 72° C. for 2 minutes was carried out 16cycles. The primers used were as follows.

TIM-3 Fw2: (SEQ ID NO: 20) 5′-GCCACCATGTTTTCACATCTTCCCTT-3′C-FLAG-NotR2: (SEQ ID NO: 21)5′-AAAAGCGGCCGCTCACTTGTCGTCATCGTCCTTGTAGTC-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. A band at around 0.6 kb was cut out andextracted using Wizard SV Gel and PCR Clean-Up System. Then 4 μL of theextracted DNA was mixed with 0.5 μL of pGEM-T Easy vector (Promega) andligated using Quick Ligation™ Kit (New England Biolabs). Regarding thetransformation, the ligation sample and a DH10B competent cell weremixed and spread on LB plate (containing X-gal and ampicillin). Insertcheck of pGEM-T Easy vector was carried out by colony direct PCR usingLA Taq (Takara Bio Co. Ltd.). Insert check of pGEM-T Easy vector wascarried out by colony direct PCR using LA Taq (Takara Bio Co. Ltd.).Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 30seconds was carried out 38 cycles and then a reaction at 72° C. for 2minutes was carried out. The primers used were as follows.

T7: (SEQ ID NO: 22) 5′-TAATACGACTCACTATAGGG-3′ SP6: (SEQ ID NO: 23)5′-CATACGATTTAGGTGACACTATAG-3′

The thus obtained PCR products were subjected to 0.8% agarose gelelectrophoresis (135 V, 15 minutes, TAE buffer). DNA was visualized byethidium bromide staining. Using a colony from which amplification ataround 0.8 kb was obtained, nucleotide sequence was determined by adirect sequencing method. As the PCR primers, T7 and SP6 were used. Aclone containing the same sequence of the coding region of GenBankaccession number NM_032782 and its plasmid DNA was extracted by theMiniprep method.

For preparation of the expression vector for expressing soluble form ofextracellular region of human TIM-3, the clone in which 5′ terminal ofhuman TIM-3 cDNA was at T7 side in cloning site of pGEM-T Easy vector.hTIM-3/GEM-T Easy plasmid DNA and pEF6Myc_HisC (Invitrogen) weredigested with NotI and subjected to 0.8% agarose gel electrophoresis(135 V, 15 minutes, TAE buffer). DNA was visualized by ethidium bromidestaining. Bands around 0.6 kb and 5 kb were cut out and extracted usingWizard SV Gel and PCR Clean-Up System, respectively. Then 4 μL of theextracted sTIM-3 cDNA was mixed with 0.5 μL of pEF6 Myc_HisC vector DNA,and ligated using QuickLigation™ Kit (New England Biolabs). Regardingthe transformation, the ligation sample and a DH10B competent cell weremixed and spread on LB plate (containing ampicillin). Insert check wascarried out by colony direct PCR using LA Taq (Takara Bio Co. Ltd.).Regarding the PCR, after a denaturation step at 95° C. for 1 minute, athree step reaction at 95° C. 15 seconds-56° C. 15 seconds-72° C. 60seconds was carried out 38 cycles and then a reaction at 72° C. for 2minutes was carried out. The primers used were as follows.

TIM-3 Fw2: (SEQ ID NO: 24) 5′-GCCACCATGTTTTCACATCTTCCCTT-3′ BGH-R:(SEQ ID NO: 25) 5′-TAGAAGGCACAGTCGAGG-3′

From a colony in which amplification at around 0.8 kb was obtained, theplasmid DNA was extracted using Miniprep method.

It was confirmed by a DNA sequence analysis that the purifiedsTIM-3-FLAG/pEF6 Myc_HisC plasmid DNA had the sequence identical to thecorresponding region of GenBank accession number NM_032782. The primersused for the DNA sequence analysis were as follows.

T7: (SEQ ID NO: 26) 5′-TAATACGACTCACTATAGGG-3′ BGH-R: (SEQ ID NO: 27)5′-TAGAAGGCACAGTCGAGG-3′

The sequence of the insert (from the following NotI recognition site tojust before NotI recognition site) was as follows.

(SEQ ID NO: 28) GGGAATTCGATTGCCACCATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGATATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGATTGGCCAATGACTTACGGGACTCTGGAGCAACCATCAGAGACTAC AAGGACGATGACGACAAGTGA(Preparation of Fusion Protein of Human Fc and Soluble Form ofExtracellular Human TIM-3 and, Soluble Form of Extracellular HumanTIM-3)

Plasmid DNAs of sTIM-3-FLAG-Fc/pTracerCMV and sTIM-3-FLAG/pEF6 Myc_HisCwere purified using QIAGEN Plasmid Maxi Kit.

HEK293F cells were used as a host cell for expression. The HEK293F cellswere cultured with shaking in FreeStyle 293 Expression Medium(Invitrogen) (37° C., 5% CO₂).

The PEI method was used in the gene introduction. Polyethylenimine(Linear, MW 25,000, manufactured by Polysciences) was weighed anddissolved in PBS (1 g/l) while adjusting to around pH 7.0 with HCl. Theobtained solution was stirred for 1 hour and then sterilized byfiltering through a membrane filter having a pore size of 0.22 μm,MILLEX-GV (Millipore). Then, 1 mg of the purified plasmid DNA was mixedwith 20 ml of Opti-Pro SFM (Invitrogen) to obtain Solution A. Solution Bwas prepared by mixing 2.5 ml of PEI solution (1 g/l) with 20 ml ofOpti-Pro SFM (Invitrogen). After solution A and Solution B were mixedand allowed to stand still for 10 minutes, and then the obtainedsolution was added to 1 L of 293F cells (1,000,000 cells per 1 ml).After six days, the cell supernatant was harvested and used for theprotein purification.

Purification of the fusion protein of human Fc and soluble form ofextracellular human TIM-3 and the soluble form of human TIM-3 proteinwere carried out by the following method. The culture supernatantcontaining the fusion protein of human Fc and soluble form ofextracellular human TIM-3 and the soluble form of TIM-3 protein wereharvested by centrifugation 6 days after the transfection and passedthrough a filter (950 ml). The obtained solution was diluted 5-fold withTris buffered saline (TBS), an anti-FLAG column was prepared usinganti-FLAG M2 Agarose Affinity Gel (Sigma) and the solution was appliedthereto using HiLoad Pump P-50 (Pharmacia Biotech). Elution was carriedout using FLAG peptide (Sigma) in accordance with the manual. The eluatewas fractionated into 8 fractions, each fraction was subjected toSDS-PAGE (MultiGel II Mini 10/20% gradient gel; Cosmo Bio Co., Ltd.)under a reducing condition, and then silver staining and Westernblotting were carried out. A silver staining reagent “Daiichi” (DaiichiPure Chemicals Co., Ltd.) was used for the silver staining. Anti-FLAG M2antibody (Sigma) and an alkaline phosphatase-labeled rabbit anti-mouseimmunoglobulin antibody were used for the Western blotting. Fractions inwhich the protein of interest was found were concentrated using AmiconUltra-4 10K (Millipore), and gel filtration chromatography was carriedout using Superdex 200 gp (GE Healthcare). After fractionation, eachfraction was subjected to SDS-PAGE (MultiGel II Mini 10/20% gradientgel; Cosmo Bio Co., Ltd.) under a reducing condition, and then silverstaining and Western blotting were carried out. A silver stainingreagent “Daiichi” (Daiichi Pure Chemicals Co., Ltd.) was used in thesilver staining. Anti-FLAG M2 antibody (Sigma) and an alkalinephosphatase-labeled rabbit anti-mouse immunoglobulin antibody were usedin the Western blotting. Fractions in which the protein of interest wasfound were concentrated using Amicon Ultra-4 10K (Millipore) and washedwith PBS. By carrying out sterilization by filtration using a membranefilter MILLEX-GV (Millipore) having a pore size of 0.22 μm, a fusionprotein of human Fc and soluble form of extracellular human TIM-3, andsoluble form of extracellular human TIM-3 were obtained. As a result ofLimulus test using Limulus ES-II Kit Wako (Wako Pure ChemicalIndustries, Ltd.), endotoxin was not detected in the fusion protein ofhuman Fc and soluble form of extracellular human TIM-3, and the solubleform of extracellular human TIM-3. Regarding concentration of the in thefusion protein of human Fc and soluble form of extracellular humanTIM-3, and the soluble form of extracellular human TIM-3, absorbance at280 nm was measured and 1 mg/ml was calculated as 1.4 OD.

Example 7 Cytotoxicity Test of TIM-3 Expressing Cell Line UsingAnti-TIM-3 Polyclonal Antibody

Regarding the cellular cytotoxicity mediated by an antibody,cytotoxicity for target cells (Antibody-Dependent Cellular Cytotoxicity,hereinafter ADCC) was measured in the presence of an antibody usinghuman peripheral blood-derived mononuclear cells (hereinafter PBMCs) asan effector.

Goat-derived anti-TIM-3 polyclonal antibody (R & D Systems; AF2365) wasused as an antibody. In addition, goat-derived IgG (manufactured bySIGMA; 15256-10MG) was used as a negative control.

Regarding the method, briefly, a target cells are cultured in thepresence of PBMCs and lysis of the target cells by an antibody ismeasured.

Specifically, as a target cell, each of TIM-3-transfected Jurkat cellsand a TIM-3-transfected EoL-1 cells were cultured with sodium chromatelabeled with a radioisotope ⁵¹Cr (Na₂ ⁵¹CrO₄, manufactured byPerkinElmer Corp., NEZ030S) at 37° C. for 1 hour in the presence of 5%CO₂ to label the target cell with ⁵¹Cr. After the labeled target cellswere washed three times with RPMI-1640 medium containing 10% FCS toremove excess ⁵¹Cr, the cells were suspended in the medium (40,000cells/ml) and then dispensed in 50 μl/well into a 96-well plate. PBMCswere suspended in the medium (4,000,000 cells/ml) and dispensed in 50μl/well into the plate (effector/target ratio=100). An antibody wassuspended in the medium and 50 μl of the obtained suspension was addedto the plate (final concentration 10 μg/ml) to culture at 37° C. for 4hours in the presence of 5% CO₂. As the antibody, anti-human TIM-3 goatpolyclonal antibody (manufactured by R & D Systems) was used, and goatIgG (manufactured by SIGMA) was used as the negative control. The PBMCs,using as the effector, were derived from healthy human peripheral blood.

Regarding the lysis rate of target cells, amount of ⁵¹Cr in the sodiumchromate released into the medium due to the lysis of cells wasmeasured. That is, after the plate was centrifuged, the supernatant wastransferred in 50 μl portions into a scintillator-coated 96-well plate(Lumaplate-™, manufactured by Perkin-Elmer Corp.) and then dried at 56°C. for 2 hours. The plate was sealed (TopSeal-A, manufactured by PackardInstrument Co., Inc.) and measured using a microplate reader (TopCount,manufactured by Perkin-Elmer Corp.).

The results are shown in FIG. 16. Regarding the significance test, bycomparing with the goat IgG using the standard Student's t-test, thosehaving a level of significance (p) of 0.05 or less was considered to besignificantly different.

In comparison with the human-derived IgG control, it was found that theanti-TIM-3 polyclonal antibody exhibited significant increase lysis rateof the target cell as shown below. For this reason, the anti-TIM-3polyclonal antibody was shown to have the ADCC on TIM-3 expressioncells. Accordingly, the example showed a possibility of treatment inwhich removal of TIM-3 positive cells using ADCC could be drug efficacy.

In addition, as cytotoxicity on target cells, Complement-DependentCytotoxicity (hereinafter CDC) was measured in the presence ofcomplement and an antibody.

Regarding the method, briefly, radioactive chrome (⁵¹Cr) wasincorporated into the cytoplasm of target cells and then the amount of⁵¹Cr released into culture medium by cell death was measured as theamount of γ ray.

Specifically, 10⁶ cells of the TIM-3-transfected EoL-1 cell orTIM-3-transfected Jurkat cell as a target cells were suspended in 15 μlof Fetal Calf Serum (FCS), mixed with 50 μl (37 MBq/ml) of ⁵¹Cr-labeledsodium chromate (manufactured by Perkin-Elmer Corp., hereinafterreferred to as ⁵¹Cr) and cultured at 37° C. for 1 hour. Next, ⁵¹Cr whichwas not incorporated into the cells was removed by repeating the step,consisting of adding 10 ml of the medium, centrifugation and discardingthe medium, three times.

Regarding CDC, 2000 cells of the ⁵¹Cr-labeled TIM-3-transfected EoL-1cell were mixed with 25% in final concentration of baby rabbitserum-derived complement (manufactured by Cedarlane; CL3441) and theantibody having each concentration (0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1μg/ml or 10 μg/ml in final concentration). In a U bottom 96-well plate,total volume of the mixed liquid was adjusted to 150 μl and cultured at37° C. for 2 hours in the presence of 5% CO₂. In addition, 0.33% infinal concentration of Triton-X100 was added thereto as a control. Afterculturing, the plate was centrifuged so as to precipitate the cells.Fifty μl of the supernatant was transferred into a powderscintillator-containing 96-well plate (Lumaplate™-96: manufactured byPackard Instrument Co., Inc.), and the plate was dried at 56° C. for 2hours. After confirmation of drying, the plate was sealed (TopSeal™-A:96-well Microplates: manufactured by Packard Instrument Co., Inc.) andthe amount of 7 ray was measured using a scintillation counter(TopCount, manufactured by Packard Instrument Co., Inc.).

A specific lysis rate was calculated by dividing a value obtained bysubtracting the γ ray dosage at an antibody concentration of 0 μg/mlfrom the value of each well, by the value of the well to whichTriton-X100 was added (specific lysis rate is regarded as 100%). Theresults are shown in FIG. 17. It was found that TIM-3 polyclonalantibody exhibited cytotoxicity on target cells inconcentration-dependent manner. Accordingly, the example showed apossibility of treatment in which removal of TIM-3 positive cells usingCDC would be drug efficacy.

Example 8 AML Primary Cell Toxicity Test by Anti-TIM-3 PolyclonalAntibody

Regarding the cytotoxicity mediated by an antibody, as cytotoxicity ontarget cells, complement-dependent cytotoxicity (hereinafter CDC) wasmeasured in the presence of complement and an antibody.

Regarding the method, briefly, target cells are cultured in the presenceof a complement and the decrease of the cell viability due to theantibody is measured.

As an antibody, goat-derived anti-TIM-3 polyclonal antibody was used,and as an antibody of negative control, goat-derived IgG (manufacturedby SIGMA; 15256-10MG) was used.

Specifically, primary AML cells from a patient as target cells weresuspended in RPMI-1640 medium containing 10% FCS and 1,000,000cells/well were dispensed into a 24-well plate (462.5 μl/well). To thecell, 12.5 μl/well (2.5% in final concentration) of baby rabbitserum-derived complement (manufactured by Cedarlane; CL3441) was added.After 25 μl of goat-derived anti-TIM-3 polyclonal antibody (0.2 mg/ml)was added (10 μg/ml in final concentration), the cells were cultured at37° C. for 3 hours in the presence of 5% CO₂.

Ratio of dead cells was evaluated by flow cytometry by AnnexinVstaining. After washing with PBS, the cells were suspended in 50 μl ofAnnexinV Binding Buffer (manufactured by BD) again, and stained at roomtemperature for 10 minutes by adding PI and AnnexinV-FITC (manufacturedby BD). After diluting with 200 μl of AnnexinV Binding Buffer(manufactured by BD), the sample was analyzed by FACSAria (manufacturedby BD). The ratio of the AnnexinV(+)PI(−) cells and PI(+) cells in theentire AML cells was regarded as the ratio of dead cells.

The results are shown in FIG. 8. Regarding the significance test, bycomparing with the human-derived IgG using the standard Student'st-test, those having a level of significance (p) of 0.05 or less wasconsidered to be significantly different.

In comparison with the human-derived IgG control, significant increasein the ratio of dead to live cells was found in the anti-TIM-3polyclonal antibody. For this reason, it was shown that the antibody hadCDC on TIM-3 positive primary AML cells. Accordingly, the example showeda possibility of treatment in which removal of primary AML cells wouldbe drug efficacy.

INDUSTRIAL APPLICABILITY

The invention can provide a method for treatment comprisingadministering anti-TIM-3 antibody to a subject which is a candidate ofblood tumor in which TIM-3 is expressed in Lin(−)CD34(+)CD38(−) cellfraction of bone marrow or peripheral blood or a subject who hasreceived treatment for blood tumor.

FREE TEXT OF SEQUENCE LISTING

-   SEQ ID NO:3: TIM-3Fw2 primer-   SEQ ID NO:4: TIM-3 Re2 primer-   SEQ ID NO:5: T7 primer-   SEQ ID NO:6: SP6 primer-   SEQ ID NO:7: pMCs-Fw primer-   SEQ ID NO:8: TIM-3 Re1 primer-   SEQ ID NO:9: pMCs-Fw primer-   SEQ ID NO:10: hTIM-3 Fw1 primer-   SEQ ID NO:11: pMCs-Fw primer-   SEQ ID NO:12: TIM3ED-FcReXba primer-   SEQ ID NO:13: T7 primer-   SEQ ID NO:14: TIM3ED-FcReXba primer-   SEQ ID NO:15: T7 primer-   SEQ ID NO:16: hTIM-3 Fw1 primer-   SEQ ID NO:17: Insert (from the following EcoRI recognition site just    prior to ApaI recognition site)-   SEQ ID NO:18: TIM-3Fw2 primer-   SEQ ID NO:19: TIM3ED-FLAG4aa primer-   SEQ ID NO:20: TIM-3 Fw2 primer-   SEQ ID NO:21: C-FLAG-NotR2 primer-   SEQ ID NO:22: T7 primer-   SEQ ID NO:23: SP6 primer-   SEQ ID NO:24: TIM-3 Fw2 primer-   SEQ ID NO:25: BGH-R primer-   SEQ ID NO:26: T7 primer-   SEQ ID NO:27: BGH-R primer-   SEQ ID NO:28: Insert (from the following NotI recognition site just    prior to NotI recognition site)

What is claimed is:
 1. A method for treating a tumor in a subject,comprising administering an anti-T-cell immunoglobulin and mucin-domaincontaining-3 (TIM-3) antibody or its TIM-3 binding fragment to thesubject who is suspected to have a tumor in which a cell expressingTIM-3 is recognized in bone marrow or peripheral blood, wherein the cellis at least one cell fraction of (a) Lin(−)CD34(+)CD38(+) or (b)Lin(−)CD34(−).
 2. The method according to claim 1, wherein the tumor islymphoma, myelodysplastic syndromes, acute myeloid leukemia or chronicmyeloid leukemia.
 3. The method according to claim 1, wherein the bloodtumor is recurrence and/or refractory.
 4. The method according to claim1, wherein the anti-TIM-3 antibody is an antibody which has a constantregion originated from a human species.
 5. The method according to claim1, wherein the anti-TIM-3 antibody is at least one antibody selectedfrom the group consisting of a monoclonal antibody, a chimeric antibody,a humanized antibody and a human antibody.
 6. The method according toclaim 1, wherein the anti-TIM-3 monoclonal antibody is an antibodyhaving ADCC activity and/or CDC activity.
 7. A method for decreasing atumor in a subject comprising administering an anti-T-cellimmunoglobulin and mucin-domain containing-3 (TIM-3) antibody or itsTIM-3 binding fragment to the subject who is suspected to have tumor inwhich a cell expressing TIM-3 is recognized in bone marrow or peripheralblood, wherein the tumor is recurrence and/or refractory.
 8. The methodaccording to claim 7, wherein the cell is at least one cell fractionselected from the group consisting of (a) to (c): (a)Lin(−)CD34(+)CD38(−), (b) Lin(−)CD34(+)CD38(+), and (c) Lin(−)CD34(−).9. The method according to claim 8, wherein the cell fraction (a) is aleukemia stem cell.
 10. The method according to claim 8, wherein thecell fraction (b) or (c) is a blast cell.
 11. The method according toclaim 7, wherein the tumor is lymphoma, myelodysplastic syndromes, acutemyeloid leukemia or chronic myeloid leukemia.
 12. The method accordingto claim 7, wherein the anti-TIM-3 antibody is an antibody which has aconstant region originated from a human species.
 13. The methodaccording to claim 7, wherein the anti-TIM-3 antibody is at least oneantibody selected from the group consisting of a monoclonal antibody, achimeric antibody, a humanized antibody and a human antibody.
 14. Themethod according to claim 7, wherein the anti-TIM-3 monoclonal antibodyis an antibody having ADCC activity and/or CDC activity.
 15. The methodaccording to claim 1, wherein the cell fraction further comprises (c)Lin(−)CD34(+)CD38(−).
 16. The method according to claim 1, wherein thecell fraction (a) or (b) is a blast cell.
 17. The method according toclaim 15, wherein the cell fraction (c) is a leukemia stem cell.
 18. Themethod according to claim 15, wherein the cell fraction is a cellfraction selected from the group consisting of (a) and (b), (a) and (c),(b) and (c), and (a), (b) and (c).